extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
A caspase-activated factor (CAF) induces mitochondrial membrane depolarization and cytochrome c release by a nonproteolytic mechanism.

It is well established that apoptosis is accompanied by activation of procaspases and by mitochondrial changes, such as decrease in mitochondrial transmembrane potential (ΔΨm) and release of cytochrome c. We analyzed the causal relationship between activated caspases and these mitochondrial phenomena. Purified recombinant caspase-1, -11, -3, -6, -7, and -8 were incubated with mitochondria in the presence or absence of additional cellular components, after which ΔΨm was determined. At lower caspase concentrations, only caspase-8 was able to activate a cytosolic factor, termed caspase-activated factor (CAF), which resulted in decrease in ΔΨm and release of cytochrome c. Both CAF-mediated activities could not be blocked by protease inhibitors, including oligopeptide caspase inhibitors. CAF-induced cytochrome c release, but not decrease of ΔΨm, was blocked in mitochondria from cells overexpressing Bcl-2. CAF is apparently involved in decrease of ΔΨm and release of cytochrome c, whereas Bcl-2 only prevents the latter. Hence, CAF may form the link between death domain receptor–dependent activation of procaspase-8 and the mitochondrial events studied.

central part in both induction and execution of apoptosis mediated by Fas and the p55 TNF receptor. Caspases are expressed as inactive zymogens and become activated by autoproteolytic processing (1) or cleavage by other caspases (2) at specific aspartic acid residues. After activation, caspases cleave their substrates, e.g., caspase zymogens, as well as cytosolic, nuclear, and cytoskeletal proteins (3). For several caspases, participation in cell death, especially in Fas-induced apoptosis, has been established (4). After aggregation of Fas and subsequent formation of the death-inducing signaling complex (DISC), recruitment of procaspase-8 to DISC is followed by autoproteolytic activation of this caspase (5,6). Depending on the amount of active caspase-8 generated at the receptor complex, two types of Fas-induced apoptosis have been described (7). In type I cells, recruitment of high amounts of procaspase-8 results in direct activation of executioner caspases accompanied by loss of mitochondrial transmembrane potential ( ⌬⌿ m ) and release of cytochrome c from the mitochondria. In type II cells, where low amounts of procaspase-8 are re-cruited to DISC, mitochondrial membrane depolarization (MMD) and release of cytochrome c precede activation of caspase-3. This release of cytochrome c may initiate the execution phase (8) by Apaf-1-mediated processing of procaspase-9 to active caspase-9, resulting in activation of caspase-3 (9,10). Accordingly, inhibition of MMD and cytochrome c release by overexpression of the antiapoptotic protein Bcl-2 blocks apoptosis in type II but not in type I cells (7).
In addition, other triggers of apoptosis, such as overexpression of the proapoptotic protein Bax (11), accumulation of the secondary messenger ceramide (12), treatment with etoposide or staurosporine (13,14), and exposure to ionizing radiation (14,15), are dependent on or accompanied by MMD and/or release of cytochrome c from the mitochondria. Recently, activation of a cytochrome c efflux-inducing factor (CIF) during staurosporine-induced apoptosis has been demonstrated (16). However, the mechanism by which death receptors induce MMD and the release of cytochrome c remains elusive. In particular, the relative contribution, if any, of the executioner caspases-3, -6, and -7, but also of the initiation caspases-1, -11, and -8, to the generation of these mitochondrial phenomena remains an intriguing question.
In this paper, we analyzed the efficacy of purified recombinant caspase-1, -3, -6, -7, -8, and -11 to induce MMD in isolated mitochondria in the presence or absence of additional cellular components. We identified an intermediary protein, termed caspase-activated factor (CAF), which generates MMD and cytochrome c release by a nonproteolytic mechanism. Accordingly, CAF may constitute a crucial link between the caspase cascade and these mitochondrial events.
Preparation of Organelles. 10 ϫ 10 6 PC60 cells were permeabilized with 0.01% digitonin in 2 ml of isolation buffer, pH 7.4, comprising 220 mM mannitol, 68 mM sucrose, 2.5 mM PO 4 H 2 K, 1 mM EGTA, 10 mM Hepes-NaOH, and 0.5% BSA. Mitochondria were isolated by differential centrifugation. After homogenization, cell debris and supernatant were removed by centrifugation (20 s at 10,000 g and 10 min at 15,000 g , respectively) to pellet down the mitochondria. The latter were resuspended in isolation buffer and stored on ice for up to 1 h. All steps were carried out at 4 Њ C.
Determination of MMD. Serial caspase dilutions were obtained in cell-free system buffer, supplemented with 5 mM dithiothreitol and 2 mM ADP. 10 5 permeabilized PC60 cells or isolated mitochondria from 10 5 PC60 cells were incubated with caspases for 30 min at 37 Њ C. MMD was detected by flow-cytometric analysis of R123 accumulation (FACScalibur ® ; Becton Dickinson, Mountain View, CA [19]). R123 was excited with a 488-nm argon ion laser; fluorescence emission was measured at 525 nm.
Determination of Cytochrome c Release. 10 6 digitonin-permeabilized PC60 cells or isolated mitochondria from 10 6 PC60 cells were incubated in cell-free system buffer, with or without caspase-8 or in caspase-8-activated or untreated cytosol extract, both supplemented with 5 mM dithiothreitol and 2 mM ADP at 37 Њ C for 30 min. Opened cells or mitochondria were pelleted by centrifugation (15,000 g for 5 min). Supernatant was analyzed by 15% SDS-PAGE, using mAB to cytochrome c (PharMingen, San Diego, CA) and ECL-based detection (Nycomed Amersham plc, Little Chalfont, Bucks, UK).

Results and Discussion
Caspase-8 Induces MMD in Cooperation with an Additional Cellular Factor. It was recently reported that caspase-1, -2, -3, -4, and -6 induce MMD in intact mitochondria (20). However, to obtain the MMD-inducing effect, high amounts of caspase activity were used. To verify whether additional cellular components promote the MMD-inducing activity at low caspase concentrations, we quantified the MMD-inducing capacity of serial dilutions from murine caspase-1, -11, -3, -6, -7, and -8 on isolated PC60 mitochondria and on mitochondria in permeabilized PC60 cells. Cells had been permeabilized with digitonin, a mild detergent which, at low concentration, dissolves plasma membranes without harming mitochondrial membranes (21). This open cell system allows the introduction of membrane-impermeable proteins while preserving the integrity of mitochondria and maintaining the presence of cytosolic components. MMD was determined by flowcytometric analysis of ⌬⌿ m using the mitochondria-specific and potential-sensitive dye, R123 (19). Induction of MMD by uncoupling of the respiratory chain with the protonophore mClCCP or by atractyloside, an inhibitory ligand of the adenine nucleotide translocator which induces MMD by opening the permeability transition pores (22), resulted in a 10-fold drop in R123 fluorescence intensity both in isolated mitochondria and opened cell mitochondria (Fig.  1, inset ). This shows that the two types of mitochondria display similar functionality and that both in vitro systems are suited for MMD analysis.
The critical concentration needed to induce MMD was determined for each caspase. In isolated PC60 mitochondria (Fig. 1), caspase-6 and -8 were highly inefficient and generated MMD at a concentration of 60 g/ml only. Caspase-1 and -3 were slightly more efficient, exhibiting a critical concentration of 30 g/ml. Caspase-11 and -7 scored best, with critical concentrations of 15 and 8 g/ml, respectively. These results are in agreement with previous reports demonstrating that caspases induce MMD in intact mitochondria (20), and further substantiate the low efficacy at which caspases, especially caspase-6 and -8, generate this response.
In contrast to the low efficacy in isolated mitochondria, all caspases showed a higher MMD-inducing activity in opened cell mitochondria (Fig. 1), the critical concentration decreasing twofold for caspase-1 and -7, fourfold for caspase-11 and -6, or eightfold for caspase-3. However, caspase-8 exhibited the most drastic shift, evolving from nearly no activity on isolated mitochondria to the most efficient activity in opened cells, with a critical concentration of 0.5 g/ml. These results indicate that the caspases assayed induce, in intact cells, MMD mainly or exclusively (caspase-8) by cooperation with an intermediate cellular component(s), rather than by a direct mechanism. Since caspase-8 is the most effective caspase for inducing MMD in permeabilized cells and also the most apical caspase activated in the Fas-induced signal transduction pathway to cell death (5, 6), such cooperative activity may combine recruitment to DISC of procaspase-8 and occurrence of MMD.

Caspase-8 Activates a Cytosolic Protein that Induces MMD in Isolated Mitochondria by a Nonproteolytic Mechanism.
To reconstitute the cytosolic signaling cascade to the mitochondria, cytosol extracts of digitonin-permeabilized PC60 cells were applied to isolated PC60 mitochondria. Without additional treatment, these cytosol extracts had no effect on ⌬⌿ m (Fig. 2). However, the addition of caspase-8 enabled the induction of MMD. The minimal caspase-8 concentration required to activate the cytosol extract was 0.5 g/ml, at which concentration caspase-8 cannot induce MMD in isolated mitochondria. This substantiates the necessity of a cooperative cytosolic factor in order to obtain induction of MMD by caspase-8 in isolated mitochondria. The new factor is a protein, since the caspase-8-activated extract retained its MMD-inducing activity after dialysis, but became inactive after heat treatment (data not shown).
We also verified whether caspase-8 or secondary activated caspases in the cytosol extract are responsible for inducing MMD or, alternatively, whether MMD is induced by a secondary noncaspase effector molecule. To that end, all caspase activity in the caspase-8-treated cytosol extract was blocked with 10 M of the broad-spectrum caspase inhibitor zVAD-fmk. Although this resulted in complete loss of caspase activity (Fig. 2, inset ), the MMD-inducing activity of the treated extract remained unaffected (Fig. 2). In addition, Ac-YVAD-cmk and zDEVD-fmk, which at 10 M are specific inhibitors of the caspase-1 and -3 subfamilies, respectively, did not block the MMD-inducing activity in the caspase-8-activated extract (data not shown). In the cytosol extract of permeabilized PC60 cells, caspase-8 apparently activates an MMD-inducing activity that does not depend on caspase activity. This suggests the existence of an additional effector protein, which induces MMD in isolated mitochondria. Because the protein is activated by caspase treatment, we termed it CAF.
Treatment of caspase-8 with zVAD-fmk rendered caspase-8 unable to generate CAF (data not shown). Thus, generation of CAF requires caspase-8 proteolytic activity, so CAF may be part of a proteolytic cascade. Accordingly, we verified whether the MMD-inducing activity of CAF itself is exerted by a proteolytic or nonproteolytic mechanism. However, protease inhibitors specific for different protease families did not affect the ability of caspase-8treated cytosol extracts to induce MMD in isolated mitochondria (Table 1). These results show the existence of a caspase-activated protein, CAF, which possibly connects procaspase-8 recruitment and activation by death receptor aggregation with the occurrence of MMD.
CAF Induces Cytochrome c Release from Isolated Mitochondria. The major proapoptotic activity of mitochondria is the release of cytochrome c from the mitochondrial intermembrane space (11)(12)(13)(14)(15), resulting in activation of downstream caspase-3 (9, 10). We show that caspase-8-activated and zVAD-fmk-treated cytosol extract (CAF) induces cytochrome c release from isolated PC60 mitochondria (Fig.  3). Untreated cytosol extracts or caspase-8 by itself were unable to induce cytochrome c release. This indicates that CAF, by releasing cytochrome c from the mitochondria, may contribute to activation of caspase-3 and to execution of the apoptotic program.
Bcl-2 Prevents CAF-induced Cytochrome c Release, But Not MMD in Vitro. It was shown in various models of apoptosis that overexpression of Bcl-2 prevents MMD and cytochrome c release (7,13,14). Using permeabilized PC60 cells overexpressing Bcl-2 as a result of gene transfection (17), we analyzed the effect of Bcl-2 on CAF-induced MMD and cytochrome c release. Mitochondria in opened neomycin-resistant control cells showed cytochrome c release after treatment with caspase-8-activated and zVADfmk-treated cytosol extract as well as MMD, confirming that CAF entered the permeabilized cells. Addition of CAF  to opened Bcl-2-overexpressing PC60 cells resulted in release of only marginal amounts of cytochrome c, whereas MMD was induced to the same extent as in neomycinresistant control cells (Fig. 4). This shows that Bcl-2 primarily blocks the CAF-induced release of cytochrome c, which is the mitochondrial event crucial for execution of apoptosis, while CAF-induced MMD remains unaffected. Conclusion. We provide compelling evidence for the existence of a cytosolic protein, CAF, which is activated by caspase-8 after proteolytic processing and which induces MMD as well as proapoptotic mitochondrial function of cytochrome c release by a nonproteolytic mechanism. Since caspase-8 by itself only induces MMD at extremely high ( Ͼ30 g/ml), hence nonphysiological concentrations, CAF is a possible component of the death receptorinduced pathway to the mitochondria. Furthermore, CAFinduced cytochrome c release was blocked by overexpression of Bcl-2, which meets the inhibitory activity of Bcl-2 on cytochrome c release and on MMD observed in several models of apoptosis (7,(12)(13)(14). However, in contrast to previous observations, overexpressed Bcl-2 did not prevent CAF-induced MMD in opened cell mitochondria. This might indicate that Bcl-2 prevents, by its effect on the mitochondrial volume homeostasis (23), the rupture of the mitochondrial outer membrane and thus the release of cytochrome c, while its effect on the presumed opening of the permeability transition pore and the resulting MMD (22) remains limited. An alternative mechanism derives from the observation that cytochrome c release precedes MMD (13)(14)(15) or occurs in the absence of MMD (24). As a consequence and despite the fact that both CAF-induced mitochondrial events occurred almost simultaneously (data not shown), cytochrome c release and MMD may represent two independent outcomes of the same CAF-induced event differentially regulated by Bcl-2.


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Glucocorticoid Receptor Transcriptional Activity Determined by Spacing of Receptor and Nonreceptor DNA Sites*

The glucocorticoid receptor (GR) displays distinct modes of regulation when bound at glucocorticoid response elements (GREs) bearing different binding sequences and arrangements of binding sites. For example, it has been shown to activate transcription synergistically with itself or with other regulatory factors, such as AP1, when bound to a consensus palindromic element or “simple GRE” that is multimerized or linked tightly with an AP1 site. In contrast, at certain “composite GREs” GR and AP1 bind to nonconsensus sequences, and GR either activates or represses depending on the subunit composition of AP1. To uncouple the contributions to regulatory behavior of binding sequences and binding element arrangements, we examined GR action at “paired elements,” combinations of a simple GRE and a consensus AP1 site, separated by different distances. We found that GR synergized with either c-Jun or c-Jun-c-Fos at paired elements with GRE-AP1 site separations of ≥26 base pairs. In contrast, paired elements with separations of 14–18 base pairs mimicked the composite GRE, i.e. GR synergized with c-Jun and repressed c-Jun-c-Fos. In DNA binding studies, GR and AP1 cooccupied the paired elements. We conclude that the arrangement of binding sites within a compound response element can be a major determinant of regulatory factor action.

Gene regulatory regions frequently encompass several kilobase pairs of DNA and contain numerous binding sites for transcriptional regulatory factors (1). The rate of transcription initiation for any particular gene is determined by a combination of synergistic and inhibitory interactions among the regulatory proteins that are brought, directly or indirectly, to the DNA (2)(3)(4)(5). In some cases, binding sites for interacting factors may be separated by hundreds if not thousands of base pairs (bp), 1 whereas in others two or more regulatory factors may bind in close proximity to each other. In yet other cases, only one of the factors may bind, whereas interacting factors are tethered to the DNA through protein-protein interactions. The glucocorticoid receptor (GR) is a hormone-dependent transcription regulator that uses all of these types of DNA sites to achieve a complex array of context-dependent regulatory effects (6, 7): 1) Synergistic activity results when binding sites for a variety of factors are paired with a consensus GR-binding site (also called a simple hormone response element (HRE)) (3); 2) GR represses the activity of certain activators such as AP1 and NF-B at their respective consensus sites (8,9). This repression does not require binding of GR to the sites that bind only the target factor and are thus termed "tethering" elements. GR activity at this type of element appears to require direct protein-protein interaction between GR and the repressed factor; 3) GR can either repress or stimulate transcription at composite GREs (cGREs). For example, the proliferin cGRE has juxtaposed binding sites for GR and AP1 family members, and the composition of the AP1 dimer bound determines whether the receptor represses or activates transcription (4, 10 -12). When AP1 is comprised of c-Jun-c-Jun homodimers, the receptor activates transcription from a linked gene, and when AP1 is comprised of c-Jun-c-Fos heterodimers, the receptor represses transcriptional activity.
Thus, the regulatory response of each of these sequences differs qualitatively from the others. The first two types are typically induced (simple and paired elements) or repressed (tethering elements) by the receptor, whereas the third, the cGRE, can activate or repress in a context-dependent fashion. Consistent with its distinct regulatory behavior, the sequence of the prototypic cGRE, plfG, is also quite different from that of the simple HRE. Only short stretches (up to 3 bp) of the consensus HRE can be discerned, whereas a near-consensus AP1 site (5 of 7 match) can be recognized (4). Both AP1 and GR bind to the element in vitro. More recently, other cGREs have been identified, also characterized by juxtaposed or overlapping GRand AP1-binding sites and the same biphasic response to GR, depending on the composition of AP1 (10,11).
In principle, there are several possible mechanisms that could underlie the distinct behavior of the cGRE. We consider here four classes: 1) The DNA could have an allosteric effect on the receptor, AP1, or both that influences their activities. This type of effect could be either sequence-driven or DNA topologydriven (see below); 2) The relative position of the factors on the DNA could introduce constraints that permit only certain interactions; 3) The DNA site could bind a third factor that specifies activation or repression; 4) The composition of AP1 could specify activation versus repression whenever DNA-binding sites for both GR and AP1 are present, regardless of spacing or sequence. According to this mechanism, GR would synergize with c-Jun and repress c-Jun-c-Fos at any element that has binding sites for both.
The above considerations led us to ask whether the sequence of the binding sites, the composition of the AP1, or the relative positions of the factor-binding sites was the critical determi-nant of composite element behavior. We therefore constructed a series of paired response elements consisting of a simple HRE at varying distances from a consensus AP1 site (see Fig. 1) and tested their transcriptional activity in the presence of GR, c-Jun, and c-Fos, as well as their ability to bind these factors simultaneously in vitro. We found that paired elements, appropriately spaced, can demonstrate cGRE behavior and thus that the relationship of DNA sites is an important determinant of transcription factor interactions. Moreover, paired elements that demonstrate composite behavior are able to bind both GR and AP1, simultaneously suggesting that the switch from activation to repression does not involve disruption of DNA binding but rather that the factors mutually influence each others' activities.

EXPERIMENTAL PROCEDURES
Construction of Paired Element Reporters-Paired element oligonucleotides, G(n)A, where n is the center-to-center distance, in nucleotides, between the two sites, were constructed by annealing singlestranded oligonucleotides containing the sequences shown in Fig. 1 (and below) with their reverse complements. The top strand sequences contained SalI restriction sites at the 5Ј ends, and the bottom strands contained XhoI sites at their 5Ј ends. The double-stranded oligonucleotides were then ligated into the vector ⌬ODCO, which had been digested with SalI restriction enzyme and treated with calf intestinal phosphatase (0.5 units for 15 min) followed by heat treatment to kill the phosphatase (70°C for 10 min). ⌬ODCO contains the Drosophila Adh minimal promoter driving expression of chloramphenicol acetyltransferase (4). Inserts were diagnosed by digestion with appropriate restriction enzymes, and then positives were confirmed by sequencing (Biomolecular Resource Center, UCSF). The same oligonucleotides were end labeled using T4 DNA kinase for gel shift experiments. Two forms of G(16)A were synthesized, both of which were tested in transfection assays. One was also used to make probe for gel shift experiments (see below), whereas the other was made with a unique BglII restriction site between the HRE and AP1 sites. It has the sequence: TCGAGTGTA-CAGGATGTTCTAGATCTGAGTCAG (HRE and AP1 sites are underlined). Larger insertions (n ϭ 39 nucleotides or more) were made by digesting this plasmid with BglII and ligating in size fractionated fragments. The fragments for insertion were generated by digesting pBluescript DNA with Sau3AI (four-base recognition sequence) followed by low melt gel isolation. Minipreps were performed, and plasmids that appeared to have desired size were sequenced.
Cell Culture and Transfection Experiments-60 -80% confluent culture plates of murine F9 cells (Cell Culture Facility, UCSF) were grown at 5% CO 2 , Rh-90%, 37°C in Dulbecco's modified Eagle's medium H-16 (Cell Culture Facility, UCSF) supplemented with 10% fetal calf serum (Life Technologies, Inc.). Cells were transfected in 6-cm plates using calcium phosphate precipitation, as described previously (13) with the following plasmids (prepared by the CsCl 2 method): 2 g of ⌬ODCObased reporter plasmid, 2 g of GR expression plasmid, 6RGR (14), and 0.1 g of ⌬6RL (␤-galactosidase plasmid) as an internal control for transfection efficiency. Expression plasmids pRSV-c-Jun and pRSV-c-Fos (a gift of Robert Tjian) were included as indicated in figure legends. When pRSV-c-Jun alone was transfected, 0.25 g of c-Jun expression plasmid were used, and when pRSV-c-Jun and pRSV-c-Fos were transfected together, 0.125 g of each were used. After 16 h at 37°C, the cells were washed free of precipitate, glycerol shocked for 90 s, and transferred to fresh medium containing 10 Ϫ7 M corticosterone (Sigma). 24 h later cells were harvested, and extracts were prepared and assayed for chloramphenicol acetyltransferase and ␤-galactosidase activities as described previously (13). Representative experiments are shown in bar graphs; values shown in the tables below graphs represent the mean fold activation (Ϯ S.E.) of four independent transfections performed on different days.
In Vitro Measurement of DNA Binding-Electrophoretic mobility shift ("gel shift") assays were performed using GR truncated derivative, X556, expressed in E. coli. The paired element oligonucleotides G(16)A and G(26)A (see Fig. 1) were end-labeled by using T4 DNA kinase (Boehringer Mannheim) and [␥-32 P]ATP (Amersham Pharmacia Biotech) and subjected to gel purification. Binding reactions were performed as described previously (15), and complexes were resolved on a 7.8% nondenaturing acrylamide gel. Reactions had 80 ng of GR, 60 ng of c-Jun, and 60 ng of c-Fos as shown in Fig. 4. Approximately 3 ng of probe with specific activity of 5 ϫ 10 7 cpm/g was present.

RESULTS
GR acts synergistically with various unrelated transcription factors such as Sp1, NF1, and estrogen receptor when their binding sites are near each other (3). However, synergy does not always result when binding sites for GR and another factor are near each other. Indeed, GR represses c-Jun-c-Fos activity at both consensus AP1 sites, to which GR does not bind, and at cGREs, to which both GR and AP1 bind. The GR-and AP1binding sites in the proliferin (4), ␣-fetoprotein (10), and papilloma virus (11) cGREs are closely juxtaposed with center-tocenter distances of less than 20 nucleotides. These observations raise the question of whether GR represses c-Jun-c-Fos in all contexts, regardless of the arrangement of their respective binding sites or if either the sequence or binding site spacing in the composite element is important for repression.
To address this question, we first asked if repression or synergy would occur when a simple HRE and an AP1 site were paired at a greater distance than in the composite elements. We chose, as a "typical" simple HRE, the sequence found 2.5 kilobase pairs upstream of the transcription start site of the TAT gene. This element has been found to mediate transcriptional activation, never repression, by GR in all contexts examined, whether in the setting of the endogenous gene or fused to various heterologous promoters (16). We juxtaposed this HRE with a consensus AP1 site (similarly mediating activation by AP1) at varying separations and placed the resulting "paired elements" upstream of a minimal promoter driving chloramphenicol acetyltransferase expression (Fig. 1). These reporters were cotransfected with GR and c-Jun or c-Jun and c-Fos together into AP1-deficient F9 cells (4).
As shown in Fig. 2A, we found that the TAT HRE separated by 39 bp from a consensus AP1 site served as a weak GRE in the absence of cotransfected c-Jun or c-Jun-c-Fos. Both c-Jun and c-Jun-c-Fos stimulated reporter activity, as described previously for consensus AP1 sites (17) and, importantly, GR stimulated both c-Jun-c-Jun homodimers and c-Jun-c-Fos heterodimers in a hormone-dependent manner. The augmentation FIG. 1. Sequence and relative positions of HRE and AP1 site in paired elements. HRE sequence is that of the TAT GRE, whereas AP1 site sequence is that of the collagenase TPA-responsive element (16,30). N, any nucleotide; NЈ, complement to N; n, center-to-center distance in nucleotides between HRE and AP1 site. The sequences of intervening nucleotides are shown under "Experimental Procedures." of c-Jun-c-Jun activity exceeded that of c-Jun-c-Fos largely because the c-Jun-c-Fos basal was higher, consistent with the greater activity of the heterodimer (4,8,18). GR action in conjunction with c-Jun-c-Fos was more than the sum of the activity of either alone and was therefore synergistic. However, it was substantially less than multiplicative, whereas GR action in conjunction with c-Jun-c-Jun was substantially more than multiplicative. Various other spacings were also tried with similar results (Fig. 2B). Notably, both G(26)A and G(31)A, which place the two sites in opposite phases, mediated hormone-dependent augmentation of both c-Jun-c-Jun and c-Jun-c-Fos. It is notable, however, that the hormone-induced activity in the presence of c-Jun-c-Fos was greater for G(26)A than for G(31)A.
This type of synergistic behavior is similar to that identified previously for GR and other activators (3) but is quite distinct from the behavior that GR had demonstrated previously at consensus AP1 sites (repression of either c-Jun-c-Jun or c-Junc-Fos) or cGREs (activation with c-Jun and repression of c-Junc-Fos) (4,8,10,19). These results indicate that GR does not repress c-Jun-c-Fos activity in all contexts but rather that either the spacing or sequence of the factor-binding sites (or surrounding sequence) must confer the repression phenotype.
The prototypic cGRE, plfG, was identified by the DNaseI footprint produced by either purified bacterially expressed GR DNA-binding domain (DBD) or full-length GR expressed in HeLa cells (4). Within plfG is a near-consensus AP1 site, which is protected by purified c-Jun or c-Jun-c-Fos in DNase I footprinting experiments. However, the plfG sequence bears little resemblance to the consensus sequence for a simple HRE (Fig.  1), and thus the relative spacing of the GRE and the AP1 site cannot be precisely determined. It is clear, however, that they are closely juxtaposed. With these observations in mind, we wanted to determine whether at smaller separations the paired elements would function as a cGRE. We first tested paired elements with a TAT HRE separated from a consensus AP1 site by 16 nucleotides. As shown in Fig. 3A, a TAT HRE separated by 16 nucleotides from a consensus AP1 site produces a response element, G(16)A, whose response to AP1 and GR is qualitatively identical to that of a cGRE: It is activated by GR in the presence of c-Jun, repressed by GR in the presence of c-Jun and c-Fos, and minimally affected in the absence of AP1 factors. Spacings of 14, 17, and 18 nucleotides also demonstrated this type of regulatory switch (Fig. 3B). Smaller sepa-  Fig. 2A. The graph shown is representative of four independent experiments with similar results; shown below the graph is the fold activation Ϯ standard error for the four experiments. B, composite regulation tolerates multiple spacings. Experiments were performed as above. c-Jun and c-Fos concentrations are as in Fig. 2B. The graph shown is representative of three similar experiments. rations down to 11 nucleotides also supported repression of c-Jun-c-Fos but only weak activation in the presence of c-Jun alone (data not shown). Identical results were obtained using a paired element with a different sequence between the TAT HRE and the AP1 site (see "Experimental Procedures"), indicating that the effect is due to juxtaposition of the two sites and not to the sequence of the intervening DNA. A(16)G (with reverse orientation from G(16)A) also gave a similar response, indicating that the respective proximities of AP1 and GR to the general transcription machinery are not important for this effect.
As a first step toward understanding the mechanism of the response of G(16)A, we examined the influence of GR and AP1 factors on each other's DNA binding. We examined first the ability of GR to co-occupy this element with c-Jun-c-Fos heterodimers and c-Jun homodimers in gel shift assays. We performed gel shift experiments using purified bacterially expressed GR DBD and full-length c-Jun and c-Fos proteins. GR DBD, rather than full-length, was used because the different retarded species could not be discerned using full-length receptor, consistent with previous reports (20,21). As shown in Fig.  4 (lanes 1 and 6), c-Jun-c-Fos and GR DBD, when incubated separately, each shifted G(16)A to distinct positions. When the three proteins were added together, a third species appeared (lane 2). The uppermost band and the receptor band were competed by cold TAT HRE (lane 3), whereas the uppermost band and the AP1 band were competed by cold AP1 competitor DNA (lane 4). Finally, all three bands were competed by cold G(16)A (lane 5). We conclude that the upper band represents DNA co-occupied by GR and c-Jun-c-Fos heterodimers. This conclusion was further supported by antibody supershift experiments using anti-c-Jun, anti-c-Fos, and anti-GR antibodies (data not shown). Similar results were obtained when c-Jun homodimers were incubated with GR-DBD (lane 7), indicating that GR-DBD and the AP1 factors are capable of co-occupying the paired element under activating as well as repressing conditions. Not surprisingly, GR-DBD and AP1 (c-Jun or c-Jun-c-Fos) were able to co-occupy G(26)A as well (data not shown).

Spacing between DNA-binding Sites as a Determinant of
Factor Activities-Our results show that the relative positions of a paired HRE and AP1 site profoundly influences hormoneinduced activity. When a site for only one of the factors is present, then the other has an inhibitory effect (mutual inhibition) regardless of whether AP1 is composed of c-Jun-c-Jun or c-Jun-c-Fos (8,18,22). 2 In contrast, when the two sites are both present but not closely juxtaposed (from 26 to at least 210 bp), they act synergistically regardless of the composition of AP1. However, when binding sites for both GR and AP1 are closely juxtaposed (separated by 14 -18 bp), they behave as cGREs. With respect to cGRE function, several points are of interest: First, the paired elements are composed of a simple HRE and a consensus AP1 site that mediate transcriptional activation when present alone or separated from each other by 26 bp or more. Second, the surrounding sequences can be altered without affecting the response. Third, multiple different paired element spacings support a composite response. Thus, our data suggest that the relative position of the factor-binding sites is a critical determinant of composite behavior.
AP1-induced Changes in DNA Topology: a Putative Regulatory Switch-When AP1 and GR DNA sites are paired but not closely juxtaposed, synergy may result from several possible mechanisms. These include cooperative recruitment of a coactivator, action at distinct rate-limiting steps in transcription initiation, or cooperative DNA binding (23). Although the mechanistic basis of synergy between GR and AP1 has not been determined, it is interesting to note that both interact physically with CREB-binding protein, and this interaction has been suggested to explain mutual inhibition at their respective sites by titration of this important coactivator (24). When sites for both are present they can, in principle, cooperate in the recruitment of CREB-binding protein, providing a plausible unifying mechanism for synergy at paired sites and mutual inhibition at single sites. However, this type of mechanism does not account for composite behavior because it does not account for the opposite responses to GR of c-Jun-c-Jun and c-Jun-c-Fos.
The simplest explanation for the composite response is that due to the proximity of the sites GR disrupts c-Jun-c-Fos (but not c-Jun-c-Jun binding), as has been suggested previously to explain GR repression of cAMP-response element-binding protein-driven transcription at an overlapping HRE and cAMP response element (25). Our gel shift experiments suggest, however, that GR and c-Jun-c-Fos heterodimers can bind simultaneously to the paired element. Because these experiments were done with partial length GR protein, we cannot rule out a role of the N or C terminus in disrupting DNA binding by c-Jun-c-Fos. However, two observations suggest that this is not the case: 1) Full-length GR and AP1 co-occupy plfG (4); 2) mineralocorticoid receptor has a similar ligand-binding domain and a substantially larger N terminus and binds with equal affinity to the same site yet does not repress AP1 activity at either plfG or G(16)A (13). 2 These arguments suggest that GR and c-Jun-c-Fos are bound simultaneously to G(16)A but that their activities are altered by virtue of their proximity to each other. With this idea in mind, we consider four possible mechanisms that could account for such a proximity-dependent effect: 1) c-Jun-c-Jun and c-2 D. Pearce, unpublished data. Jun-c-Fos could cause distinct local changes in DNA topology that are transmitted to GR bound nearby. Interestingly, there is evidence to support the idea that c-Jun-c-Jun homodimers and c-Jun-c-Fos heterodimers bend DNA in opposite directions, in vitro (26,27). Although the question of DNA bending by AP1 remains unresolved (28), it is appealing to speculate that the distinct local topologies have differential allosteric effects on GR conformation and activity; 2) GR, which has also been found to bend DNA (29), could exert local effects on DNA topology that influence c-Jun-c-Fos activity differently from that of c-Jun-c-Jun; 3) GR bound to the paired element might make specific base contacts with the AP1 site, resulting in a sequence-driven (as opposed to a local topology-driven) conformational change in GR as proposed for Oct1-VP16 activity at TAATGARAT sequences; 4) Constraints imposed by their proximity when bound to adjacent sites might force the GR-c-Junc-Fos complex (but not the GR-c-Jun-c-Jun complex) into an inactive conformation that, for example, interacts poorly with coactivators or the general transcription machinery.
In view of the multiple paired element spacings that are tolerated, it seems unlikely that GR is contacting the AP1 site. Rather, mechanisms based on either a factor-driven change in local DNA topology or constrained protein-protein interaction seem more likely. These latter two possibilities cannot be distinguished by our data. In principle, experiments using reporters driven by paired elements separated by intrinsically bent DNA (26) should be able to distinguish these mechanisms. Such studies have been initiated, but preliminary results have been equivocal. However, a local topology-driven DNA allosteric effect is still quite plausible and presents an intriguing extension of the concept of DNA-induced allosteric effects on transcription factor activity (6). Perhaps, by exerting distinct effects on local DNA topology, c-Jun-c-Fos heterodimers and c-Jun-c-Jun homodimers differentially influence the allosteric effect of DNA on GR activity. In any case, whether we attribute these findings to changes in protein-protein interaction or to alterations in local DNA topology, we conclude in either case that local DNA architecture plays an essential role in controlling transcription factor activities.


Forging new perspectives on disease surveillance.

Levels of Ag8 in cultural media varied considerably depending on periods of cultivation of bacteria. Additionally, the level of Ag8 varied among strains of B. pseudomallei and B. mallei. Among 61 strains of B. pseudomallei from the museum collection (most of which were isolated in Southeast Asia and northern Australia), three had increased ability to produce Ag8. These strains had been isolated from clinical specimens (blood, abscesses of hospitalized melioidosis patients) in Vietnam. The strains gave results typical of B. pseudomallei species in all routine serologic tests (agglutination test, immunofluorescence assay, immunodiffusion test). In contrast, the B. pseudomallei glanders agent (16 strains from the museum collection) had reduced ability for Ag8 production; ELISA titers of Ag8 were a thousandfold less in culture fluids in these strains. The ELISA technique not only facilitates diagnosis of disease but also provides a rational basis for selecting strains for vaccine production. It also has considerable utility for studying the pathogenicity of B. pseudomallei.

Vol. 4 To the Editor: Recognizing disease emergence as a paradigm uniquely influenced by human activity demands a reevaluation of traditional disease surveillance systems. Part of a surveillance program should be focused on the areas of human activity where disease emergence is most likely to occur. A system that monitors areas known to be involved in disease emergence, such as development projects, agriculture, climate, and refugee movements, may greatly increase our ability to detect and prevent outbreaks.
Large development projects entail ecologic upheavals that can facilitate disease emergence. Construction of a dam in 1987 in Mauritania resulted in increased mosquito breeding sites and in an explosion in the mosquito population; epidemics of Rift Valley fever quickly followed (1). The Southeastern Anatolia Irrigation Project on the Euphrates and Tigris Rivers in Turkey, which will provide irrigation for 1.7 million hectares, has already increased malaria and leishmaniasis cases in the local population (2). The massive Three Gorges Dam Project on the Yangtze River in China, which will create a reservoir 760 km long, must be evaluated for its impact on local disease. With knowledge of endemic diseases and their reservoirs and vectors in these areas of ecologic change, public health workers can anticipate disease epidemics and implement prevention measures.
Incorporating climate predictions into a disease surveillance system would supplement resources in an area known to affect disease emergence. The U.S. Agency for International Development's (USAID) Famine Early Warning System monitors the African continent for two major factors implicated in emergence: temperature and precipitation. Focused on countries at high risk for food shortages and famine, the early warning system is an example of a predictive and preventative surveillance system. Precipitation, temperature, and plant health data from satellites are evaluated as indicators of crop failure. These data are supplemented by information from field representatives who directly observe agricultural production. USAID's system and other global monitoring systems can provide a base level of surveillance that can add to our knowledge of climatologic influence on disease emergence.
The beginning of the Zairian refugee crisis in 1994 illustrates the need for surveillance among refugee populations. In July 1994, 500,000 to 800,000 Rwandan Hutus fled into the North Kivu region of Zaire. In the month between July 14 and August 14, 48,347 of these refugees died, Recognizing human involvement as a common critical factor in emergence creates the possibility of refining international disease surveillance. The Centers for Disease Control and Prevention, the World Health Organization, and national governments should foster relationships with organizations already placed to provide disease emergence information in populations and locations implicated in disease emergence. These relationships will increase the scope and efficiency of our efforts to prevent human disease.

Provide a Context for Disease Emergence
To the Editor: When a disease emerges, the trend is to assume that another important and spreading infection is about to devastate humans or animals. Some qualification of the term "emergence" is needed to put emerging diseases into a context for each target species. There may be a cause for alarm and further action or, alternatively, no real change except in knowledge. In Australia, for example, an old disease "emerged" in a new area, while in another, a disease new to the continent emerged. The two disease agents were Ross River virus (which causes fever and polyarthritis in humans) and bluetongue virus (which often causes fatal disease in sheep). Both causative viruses are insect-borne.
Ross River virus is probably of very ancient lineage as an infection transmitted between marsupials and indigenous mosquitoes and was on the Australian continent long before humans first entered (some tens of thousands of years ago). In 1975, the infection was not known to occur in Tasmania, the state separated from the Australian mainland by a wide stretch of sea. In that year, my group detected a clear-cut seroconversion to Ross River virus in sentinel cows in northern Tasmania (1). Cooperative investigations found antibody-positive sera first in marsupials and then in persons who had never left Tasmania. The existing clinical condition of polyarthritis was linked to Ross River virus only after the causative virus was recognized indirectly (2). The marsupial populations of mainland Australia and Tasmania were continuous until the seas rose at the end of the last ice age. Ross River disease had emerged in Tasmania, but only from obscurity.
Bluetongue viruses occur widely in southern and eastern Asia (3). This general picture has been established only since the discovery of bluetongue virus in Australia. Overt disease occurs in sheep on the fringes of the endemicdisease region and in susceptible sheep imported into various countries within the region (3). In


text:
A Membrane Setting for the Sorting Motifs Present in the Adenovirus E3-13.7 Protein Which Down-regulates the Epidermal Growth Factor Receptor*

The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.

A 13.7-kDa protein expressed from the "Early 3" genome region of adenovirus (E3-13.7) down-regulates the EGFR 1 and related tyrosine kinase receptors during infection of epithelial cells and fibroblasts (1,2). For EGFR it has been shown that E3-13.7 exerts its role by altering sorting at the endosomal level (3) such that the ratio of receptor destined for lysosomal degradation relative to receptor destined for recycling to the plasma membrane surface is increased. The down-regulatory action of E3-13.7 is believed to be associated with the constitutive recycling pathway of EGFR and does not require ligand stimulation of the receptor to be operative. An enhanced understanding of the down-regulatory mechanism of E3-13.7 is desirable because of light it will shed both upon the cell biology of protein trafficking pathways and upon the molecular biology of adenoviral infections.
As outlined in Fig. 1, E3-13.7 is an integral membrane protein comprised of an N-terminal transmembrane helix followed by an extracellular/lumenal loop, a second transmembrane segment, and a C-terminal cytoplasmic domain (1). While the process by which E3-13.7 down-regulates tyrosine kinase receptors is incompletely understood, it is known that the Cterminal cytoplasmic domain (E3-CyD) is critical to its function. Within this 23-residue domain there are three different sequence motifs (Fig. 1) known to be broadly relevant to the cellular trafficking of membrane proteins via clathrin-mediated internalization pathways (4 -6). One of these is the "dileucine" (87-LL) motif (Refs. 7-9 and references therein). While a functional role for this motif in E3-13.7 has yet to be established, it is of probable significance because it and adjacent residues (LLRIL) are also present in the juxtamembrane cytoplasmic domain of EGFR where it has been shown to be critical to proper sorting of the receptor. 2 The other two motifs present in the E3-CyD represent the tyrosine-based motifs YXXZ and NPXY (i.e. 72-YLRH and 76-HPQY), where Z is a large hydrophobic amino acid and X is any residue. These motifs are also critical to the internalization and intracellular trafficking of a variety of other membrane proteins and are known to be associated with clathrin adaptin recognition and binding (reviewed in Refs. 8 and 10). Carlin and co-workers 3 have established that mutation of either tyrosine to alanine in E3-13.7 leads to a loss of EGFR down-regulatory activity in adenovirus-infected cells. They have also shown that the 72-YLRH motif is required for high affinity binding of the adaptin AP-2 by the E3-CyD in vitro while both tyrosine motifs are critical for binding by AP-1.
In this paper, the conformation of the cytoplasmic domain of E3-13.7 is examined. Its structure is of relevance both because of specific insight it may provide into the mode of receptor down-regulatory action by E3-CyD and thereby into mechanisms of endosomal receptor sorting. More generally, structural information for E3-CyD may shed light upon the general mechanisms by which sorting motifs are recognized by components of cellular protein trafficking networks. While sorting motifs have previously been subjected to structural character-ization (12)(13)(14)(15)(16)(17)(18), the present study extends previous work in two crucial aspects. First, because the E3-CyD contains three different sorting motifs within a fairly short sequential span, this study will provide insight into the structural consequences of sorting signal multimerization. Clustering of signals is relevant to E3-13.7 function 3 and has been observed to be important in the sorting of other proteins (7,9). Second, most previous studies have focused on the structures of motif-containing polypeptides in aqueous solution. However, since sorting motifs are often found in sequentially juxtamembrane domains (as for E3-13.7) we considered the possibility that membrane interactions might sometimes play an important role in defining the local structure of a motif-containing segment.
In this paper we describe structural studies of these two peptides in both aqueous solution and in aqueous detergent micelles. Micelles were used to model biological membranes. The detergent used in most of our studies was dodecylphosphocholine (DPC, see Ref. 19). DPC has the same zwitterionic phosphocholine head group as the dominant phospholipid species of most eukaryotic membranes, phosphatidylcholine (20). DPC micelles have also been recently demonstrated to provide a favorable balance between desirable NMR properties and biochemical compatibility with membrane proteins, properties which are often at odds with each other (21).

EXPERIMENTAL PROCEDURES
Sample Preparation-The two peptides illustrated in Fig. 1 were obtained from Quality Controlled Biochemicals Inc. (Boston, MA). Both peptides were specified to be Ͼ95% pure according to the company documentation, but we found that the myristoylated peptide, E3-mCyD required further purification. This was accomplished by size-exclusion chromatography using Bio-Gel P2 (Bio-Rad) and water with 20% isopropyl alcohol and 0.5% formic acid as an eluant. E3-CyD powder was dissolved either in an aqueous buffer or was premixed with detergents DPC and/or SDS in perdeuterated form from Cambridge Isotopes Lab (Andover, MA). E3-mCyD peptide had a high tendency to aggregate in water and required sonication to be dissolved in the presence of micelles.
NMR Methods-NMR experiments were carried out using a Varian UNITY-Plus 600 MHz NMR spectrometer equipped with Varian 5-mm 1 H/X and 1 H/ 13 C/ 15 N indirect z axis pulsed field gradient probes. For most of the samples, the following experiments were executed: onedimensional 1 H temperature-variation, DQF-COSY (double-quantumfiltered) (22), TOCSY (23), and NOESY (using pulsed field gradients for water suppression) (24). The TOCSY mixing time was 60 -70 ms. Several NOESY mixing times were used for micellar samples: 80, 200, and 300 ms, while for detergent-free samples a single NOESY mixing time of 200 ms was used. Translational diffusion coefficients were measured using the water-suppressed longitudinal eddy-current delay pulse sequence as described by Altieri et al. (25).
J-Coupling Determination-The method of Kim and Prestegard (26) was utilized which allows accurate calculation of scalar coupling from measurement of separations of extrema in dispersive and absorptive plots of rows through cross-peaks. This method is effective in analyzing spectra with moderate-to-high signal-to-noise ratios. Additionally, traces through the antiphase DQF-COSY cross-peaks were deconvoluted with two antiphase Lorentzian resonances using VNMR. Coupling constants derived by these two methods were compared and only values which were determined to be in agreement were accepted.
Determination of Peptide Partition Coefficients from Diffusion Measurements-The NMR-based method used in this work to determine the partition coefficient of the peptides in micelles is similar to that described elsewhere (27)(28)(29)(30). In the present case, a pulsed field gradient longitudinal eddy-current delay experiment was used instead of the stimulated spin-echo experiment.
Circular Dichroism-CD spectra were acquired using a Jasco-600 spectrometer. Samples for far-UV studies were prepared by dissolving about 0.5 mg/ml peptides powder in 0 -320 mM dodecylphosphocholine (Anatrace, Maumee, OH) in buffer. The exact concentration was determined by measuring the solution absorbance at 280 nm using a calculated extinction coefficient of 2840 [M Ϫ1 cm Ϫ1 ] for both peptides. Quartz cuvettes with a light path-length of 1 mm were used. The parameters employed were: bandwidth ϭ 1 nm; sensitivity ϭ 50 mDeg; time constant ϭ 0.5 s. Spectra acquired represented an average of 4 scans. Reference spectra, obtained with the samples containing all components except the peptide, were recorded before each spectrum and subtracted from the peptide spectrum during acquisition. All spectra were recorded in mDegree units. The instrument was routinely calibrated with an standard solution of recrystallized (1R)-(Ϫ)-10-camphorsulfonic acid, ammonium salt at 290 nm ( ϭ 7910).

RESULTS
The structures of E3-CyD and E3-mCyD were examined in aqueous solution and in aqueous micelles. Measurements were made at both pH 4.5 and 7.5 for two reasons. First, this allowed data to be taken at pH above and below the pK a of the histidine side chains present in both tyrosine sorting motifs, thereby allowing us to investigate whether the structure of E3-CyD is sensitive to the state of protonation of these residues. Second, while studies at the higher pH might be more physiologically relevant, it is much easier to extract structurally useful NMR data at mildly acidic pH where all amide protons can be directly observed due to depressed rates of chemical exchange with water protons.
Structure of E3-CyD in Aqueous Solution-Total backbone 1 H NMR resonance assignments were made for E3-CyD in water at 4, 15, and 25°C at pH 4.5 and 4°C for pH 7.6. One-dimensional proton and CD spectra were routinely used as qualitative structural indicators while NOE and J-coupling measurements were used as a source of more quantitative It was observed that E3-CyD in water at 25°C is mostly unstructured at both acidic and neutral pH. Only by lowering the temperature to 4°C at pH 4.5 were we able to detect some residual ␣-helical/turn structure as reflected by d NN (i, iϩ1) cross-peaks in the NOESY spectrum ( Fig. 2A) and negative intensity at 220 nm in the CD spectrum (Fig. 3A). Even in this case, the helical content appears to be minimal. Few intermediate and no long-range NOEs were observed. Moreover, while strong sequential d ␣N (i, iϩ1) connectivities were present there was a lack of deviations by chemical shifts from their randomcoil values for C ␣ H or NH resonances ("fingerprint region" Fig.   4A). Vicinal J N␣ coupling constants fell mostly inside the range typical for extended random coil conformations. Taken together, these results indicate the lack of a defined conformation for this peptide in water under any conditions examined.
Affinity of the E3 Cytoplasmic Domain for Membranes-When E3-CyD was dissolved in DPC micelles it showed a major change in its conformational state. Chemical shifts were much more dispersed and a dramatic increase in line widths was observed relative to the peptide in solution at the corresponding temperatures (Fig. 5). The amide proton frequency of Arg 1 is shifted about 0.8 ppm downfield while amide proton frequencies of several of the C-terminal residues (Arg 21 , Ile 22 , and Leu 23 ) shifted about 0.5 ppm upfield. CD data show pronounced ␣-helical character at both acidic and neutral pH (Fig.  3A). Increasing the DPC concentration from 10 to 320 mM did not result in changes in the CD spectra (data not shown), indicating that in this range both the percentage of peptide associated with micelles and the structure of the bound peptide are independent of the peptide:micelle ratio.
Diffusion coefficients were measured which established that E3-CyD is tightly bound to DPC micelles in the 4 -40°C range and at both acidic and neutral pH ( Table I). The partition coefficients determined for the peptide under various conditions were in all cases Ͼ500 (Table I). This result indicates that the transmembrane domain of the E3-13.7 is not necessary for tight association of the cytoplasmic domain of E3-13.7 with a membrane surface. However, in order to test whether a transmembrane anchor may play an important role in defining the conformation of the surface-associated C-terminal domain, we examined an N-myristoylated form of the peptide (E3-mCyD, Fig. 1).
The E3-mCyD peptide was not water soluble. Even in micelles, it had a high tendency to aggregate, as exhibited by appreciable line-broadening. Addition of negatively charged detergents (up to 10 mol % SDS) to mimic the net negative charge present in most biological membranes did not have much affect on the spectrum. CD spectra of the myristoylated peptide were very similar to that of E3-CyD in micelles at both pH values (Fig. 3B) and the distribution of chemical shifts in its 1 H NMR spectrum was very similar to that of E3-CyD under similar conditions (not shown). These results indicate that E3-mCyD adopts a similar surface-associated structure as E3-CyD and that the membrane-associated structure of the cyto-plasmic domain of E3-13.7 can be independent of an N-terminal membrane anchor.
We also examined the question of whether E3-CyD binds to a "real" lipid bilayer with the same high affinity as DPC micelles and whether it adopts the same structure in both systems. We therefore examined the NMR spectrum of E3-CyD when mixed with unilamellar POPC vesicles in the liquid crystalline phase. POPC is a major component of eukaryotic membranes and vesicles comprised of this phospholipid are often used to mimic biological bilayers (20). The NMR spectra of E3-CyD in POPC vesicles are similar to spectra of the peptide in micelles but with much larger line widths in the former case (data not shown). The breadth of the lines prohibits detailed structural characterization in vesicles, but the general similarity of the one-dimensional spectra confirms that the peptide does indeed have a high affinity for actual lipid bilayers and strongly suggests that its structural state in bilayers is similar to its structure in micelles.
The results of this section established that E3-CyD binds with high affinity to DPC micelles in a manner which appears to be similar to its interaction with bilayers and which is independent of the presence of either an N-terminal membrane "anchor" or of a net negative charge in the interface. We therefore proceeded to a detailed structural characterization of E3-CyD in DPC micelles.
CD and NMR Structural Measurements for E3-CyD Associated with DPC Micelles-Some general structural information can be gleaned from temperature-induced changes in the am-  ide-aromatic region of one-dimensional 1 H NMR spectra of micellar E3-CyD (Fig. 5). At 4°C the spectrum is extremely broad and more highly dispersed than in the absence of detergent. When the temperature of the micellar sample is increased, peaks become sharper and less dispersed, suggestive of a loss of structural order. Some peaks undergo more changes than others in terms of both line width and chemical shift. For example, aromatic peaks of Tyr 4 at 4°C are much broader and upfield-shifted than at 40°C, while resonances from Tyr 11 are narrow at both temperatures and exhibit no changes in chemical shift. The same is true to a lesser extent for His 7 as compared with His 8 . This suggests that in the ordered (low temperature) structural state Tyr 4 and His 7 side chains are inserted into the interface while Tyr 11 and His 8 can undergo less restricted motion in the aqueous domain of the interface. Diffusion measurements indicated that both ordered and disordered states represent E3-CyD bound to the micelle surface. CD spectra indicated that there is little change in helicity over the range of 4 -40°(data not shown), suggesting that the two states may actually be conformationally similar, but have different modes of membrane interaction. The experiments described above indicate that what is observed spectroscopically for micellar E3-CyD at all temperatures is the average of two populations (ordered and disordered) in fast exchange. Unfortunately, the sample in which the ordered form is most highly populated (at 4°C) cannot be readily accessed by two-dimensional NMR experiments due to extensive line broadening. Even at higher temperatures only modest DQF-COSY and TOCSY spectra were obtained, with some spin systems missing and some peak intensities low due to line broadening. As a result, it was not possible to assign all side chain protons. The presence of two populations in fast exchange also made it hard to quantify NOE data for the ordered state and only a few 3 J N␣ coupling constants could be unambiguously measured. Nevertheless, backbone assign-ments were made at 40°C at both pH values and, while quantitative structure calculations were unfeasible, it was possible to determine a semiquantitative structural model for the ordered form of the peptide based upon these data. Fig. 2B summarizes NOE and J-coupling data obtained for E3-CyD in DPC at 40°C and acidic pH from two-dimensional NMR experiments. There are a number of intermediate-range NOEs representing the ordered population, typical for ␣-helical like structure between residues 1-6 and 17-21 NN (i, iϩ1)). The deviation of ␣-proton chemical shifts (␦H ␣ ) from their random coil values and measured vicinal J N␣ couplings are consistent with modeling these regions as ␣-helical. This indicates that 11 of 23 residues are ␣-helical, in good agreement with the 4°C CD data (Fig. 3A) estimate of about 40%. The following NOEs were also observed: Pro 9 ␦␦Ј-His 8 NH, Pro 9 ␦␦Ј-Gln 10 NH, Pro 9 ␤␤Ј-Tyr 11 NH, Pro 9 ␤␤Ј-Gln 10 NH, and Pro 9 ␤␤Ј-Gln 10 side chain NH 2 . These are indicative of a loop or ␤-turn involving residues His 8 -Tyr 11 . Furthermore, His 7 and His 8 exhibit amide-water exchange peaks, indicating that they are water accessible. Measurements at 25°C yielded data which generally confirmed the structural conclusions gleaned from the 40°C data although spectral quality at 25°C was not as high as at 40°C.
An additional source of information on the structure of the bound peptide is differential broadening of resonances from various peaks seen in the DQF-COSY and TOCSY spectra. Even at 40°C, some side chain peaks (such as those from Ile 2 , Asp 13 , Ile 16 , and Thr 15 ) are so broad that they cannot be observed, while others (Ala 3 , Tyr 4 , Ala 17 , and Asp 18 ) are clearly more broad than others. The most probable explanation is that the side chains of these residues are imbedded in the micellar interface resulting in shorter transverse relaxation times due to restrained local motion.
Increasing the pH to 7.6 did not significantly change the pattern of the NOESY fingerprint region (Fig. 4, B and C). However, some amide proton frequencies shifted at residues (in decreasing order) Thr 15 , Ile 16 , Arg 14 , Gln 10 , Ala 17 , Leu 19 , and His 8 without dramatic changes of intermediate-range NOE pattern. The major difference in the NOE pattern (data not shown) was a reduction in intensity of NN-cross-peaks for Arg 6 -His 7 , Leu 20 -Arg 21 , and Asp 18 -Arg 21 , suggestive of a population decrease for the ordered state versus the disordered one. This conclusion is supported by the CD spectrum showing a lower helical population at the higher pH. These results suggest that changing the pH does not change the overall structure of the peptide, but rather changes the stability of the ordered conformation.
Identification of Amphipathic Helices in Micelle-associated E3-CyD-Additional insight into the probable mode of the peptide-surface interaction was provided by mapping the distribution of the hydrophobicity in the helical segments. We examined helical wheel diagrams of E3-CyD to identify possible amphipathic helixes in the peptide which would be likely sites of insertion into the micellar interface. These diagrams (not shown) indicated that regions 1-7 and 17-21 are certainly amphipathic if helical. The residues comprising the hydrophobic faces of these helices correspond to the side chain resonances which exhibited the most broad NMR resonances (above section). According to this model, the first (YLRH) and the third (LL) sorting motifs exist in surface-inserted amphipathic helices, while the second one (HPQY) forms a solvent-exposed turn or loop. This model is equally valid under both mildly basic and mildly acidic conditions, is independent of the presence of a net negative charge on the micellar surface, does not require an N-terminal hydrophobic anchor, and likely can be extended to the peptide when bound to a true lipid bilayer.

Structure of Micelle-associated E3-CyD-Based
The Membrane Context of the Sorting Motifs of E3-CyD-Based on previous studies of sorting motifs in solution (12)(13)(14)(15)(16)(17)(18) it is not surprising that the 76-HPQY motif was observed to adopt a turn or loop structure although it is a little surprising that the 72-YRLH and 87-LL motifs were observed to be helical. Even more remarkable is the observed high affinity of E3-CyD for a membrane-like interface and the intimate placement of 2 of the sorting motifs within the interface and placement of the other (76-HPQY) just above. The fact that the 72-YRLH and 87-LL motifs exist within helices is a result of membrane interactions (compare with random coil results for the peptide in water). While the residue 1-7 and 17-21 segments containing these two motifs might have been predicted to adopt short amphipathic helical structures, these helices are sufficiently short such that it is unlikely that any a priori sequence-based modeling would have predicted the E3-CyD peptide to exhibit such a high affinity for a membrane-like surface in the absence of a hydrophobic anchor.
It has sometimes been observed that residue spacing between the transmembrane domain and the sorting motifs present in the juxtamembrane cytoplasmic domains of membrane proteins is critical to recognition of the motif by sorting molecules such as adaptins (10,31): removal or addition of residues from the linking region can disrupt normal sorting. While such experiments have yet to be carried out with the intact E3-13.7, it is interesting to note that if the cytoplasmic domain within native E3-13.7 does indeed lie on the membrane surface then there must be a roughly 90°conformational turn at the linkage between the transmembrane domain and the cytoplasmic domain in the native protein. Addition or removal of additional residues to this linkage could profoundly affect the structure and/or membrane interactions of the cytoplasmic domain in a manner which might have little to do with vertical spacing from the membrane surface, representing instead the perturbation of an important conformational linkage.
Implications for E3-13.7 Recognition-It is now known that the 72-YLRH motif of E3-13.7 is critical to high affinity binding of the protein by the clathrin adaptin AP-2 and that both 72-YLRH and 76-HPQY motifs are critical to AP-1 recognition and binding. 3 The affinity of the adaptins for these motifs in native and mutant form within the E3-CyD correlates with the efficiency of EGFR down-regulation by the corresponding E3-13.7 mutants in vivo. Therefore, interaction of these adaptins with the tyrosine motifs are critical to E3-13.7's in vivo function, probably to ensure appropriate trafficking of the viral protein to the endosomes. Once in endosomes it is quite possible that these or other adaptin-like molecules also interact with the sorting motifs as part of the process by which the viral protein perturbs normal receptor sorting.
The close degree of membrane association we observed for E3-CyD suggests three possible modes by which adaptins and possibly other molecules recognize the sorting motifs present in E3-CyD. First, it is possible that the sorting motifs of E3-13.7 are recognized and bound only during low probability excursions of the cytoplasmic tail off of the membrane surface and into solution. This seems improbable because of its inefficiency and because it ascribes no functional significance to intimate membrane association. A second possibility is that the sorting motifs are directly recognized at the interface. This cannot be ruled out, although one might then guess that the adaptins would be integral membrane proteins, which they are not (4). A third possibility is that sorting molecules may not recognize the sorting motifs of the cytoplasmic domain when intimately surface-associated, but that some event can occur in vivo which results in translocation of the domain off of the surface and into solution where it could then be recognized. The sort of "events" which could be imagined to bring about such a change might be post-translational modification of the domain (for which there is presently no evidence, see Ref. 1), modulation by an unidentified membrane protein, or a dramatic shift in the tilt, conformation, or membrane placement of the flanking transmembrane domain resulting in dissociation of the cytoplasmic domain from the surface. This is an intriguing possibility because it would suggest a transmembrane regulatory mechanism governing key events of E3-13.7's perturbation of normal receptor trafficking. Further experimental work will clearly be needed to establish which of the above possibilities is closest to reality, but the power of structural information to generate experimentally testable hypotheses is hopefully evident.
Implications for the Sorting of Other Membrane Proteins-In general, it is now well appreciated that the recognition of sorting motifs with membrane proteins by components of the cellular protein trafficking machinery is often dependent upon local contextual factors such as the residue spacing between a given motif and an adjacent transmembrane domain or the presence of certain residues sequentially adjacent to the sorting motif (9,10,31). The present study provides a case study in the structural consequences of motif context. For E3-CyD the fact that the sorting motifs are intimately placed near or at the membrane interface is a function of the overall affinity of the polypeptide for a membrane surface. This affinity arises not only from the residues present in the sorting motifs, but also from hydrophobic residues adjacent to the motifs: it is unlikely that either the 72-YLRH or 87-LL motif would bind tightly to a membrane surface if flanked on both sides by polar residues. Furthermore, the helical secondary structure observed for both the 72-YLRH and 87-LL motifs is almost certainly highly coupled to the fact that these motifs are set within the context of longer amphipathic helices.
How likely is it that the membrane affinity and structural characteristics observed for the E3-CyD are mirrored for sorting motif-containing segments of the many other membrane proteins containing such motifs? We examined this question by carrying out some very simple helical wheel modeling to see if NPXY, YXXZ, and LL motifs present in other proteins may also exist within longer amphipathic helices. As summarized in Table II, some but not all LL and YXXZ motifs are potentially part of amphipathic helices while NPXY is almost never flanked by a putative amphipathic helix (as for E3-13.7). These simple observations suggest that the structural conclusions drawn for E3-CyD should not be overgeneralized. Instead, the results for E3-CyD would appear to indicate that membrane affinity and placement of sorting motifs represents one of multiple possible means by which such motifs can be endowed with local structural context allowing for highly protein-specific sorting behavior in a given cell under given conditions.