Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3931-3935, April 1994 Cell Biology Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A (peptidyl- cis-trans / folding/molecular chaperone) E. ROYDON PRICE*t, MINGJIE JIN*, DAVID LIM*, SUSMITA PATI*, CHRISTOPHER T. WALSHt, AND FRANK D. MCKEON* Departments of *Cell Biology and tBiological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115 Contributed by Christopher T. Walsh, January 11, 1994

ABSTRACT Cyclophilin B is targeted to the secretory chaperone has come from in vitro studies. pathway via an signal sequence. We Cyclophilin acts early in the folding of carbonic anhydrase to analyzed the localization and trafficking of endogenous and prevent aggregation by binding to exposed hydrophobic transfected cyclophilin B in mammalian cells. Cyclophilin B domains. Only later in the folding process does cyclophilin- accumulates both in the endoplasmic reticulum and in com- mediated proline isomerization become important (15). plexes on the plasma membrane. The immunosuppressant Like the heat shock family of , the cyclophilin cyclosporin A specifically mobilizes cyclophilin B from the family of proteins contains a conserved core domain flanked endoplasmic reticulum, and promotes the of cyclo- by variable N and C termini (16). These variable domains philin B into the medium. We suggest that cyclosporin A presumably encode subcellular targeting information. While competes with endogenous plasma membrane proteins for cyclophilin A is cytosolic, cyclophilins B, C, and ninaA association with cyclophilin B in the secretory pathway. These possess cleavable ER signal sequences and are directed to the findings argue in favor ofa role for cyclophilin B as a chaperone secretory pathway (4, 17-19, 32, 39, 51, 52). In fact, the to proteins destined for the plasma membrane, rather than secreted form of cyclophilin B (CyPB) lacks the N-terminal solely as a proline isomerase functioning within the endoplas- signal sequence (17, 18). Mitochondrial and plasma mem- mic reticulum. brane cyclophilins have also been described (19, 20). Finally, CsA receptors have been reported on the surface of lympho- cytes (21). Thus the cyclophilins function in many compart- The cyclophilin proteins are highly conserved peptidyl-prolyl ments of the cell. cis-trans (PPIases) that act as intracellular re- To examine the localization and function ofCyPB, we have ceptors for the immunosuppressant cyclosporin A (CsA) (1, expressed this protein in mammalian cell lines. CyPB appears 2). CsA causes immunosuppression by forming a complex in the ER, Golgi complex, patches on the cell surface, and in with cyclophilin A that inhibits , a the medium. CsA binding specifically accelerates the traf- essential for T-cell activation (3-5). Despite this fundamental ficking of CyPB through the secretory pathway. Rather than advance in understanding T-cell suppression by CsA, the forming complexes on the cell surface, CsA-CyPB com- natural function ofcyclophilins in the cell remains unknown. plexes are secreted. These findings support the idea that Cyclophilin's PPIase activity argues for active involve- CyPB functions at all steps of the secretory pathway, pos- ment in protein folding (6, 7). This idea was supported by the sibly as a chaperone to certain proteins destined for the demonstration that cyclophilins catalyze rate-limiting proline plasma membrane or export. isomerization steps in RNase T1 and peptide folding in vitro (8, 9). Additionally, CsA blocks the PPIase activity of cy- clophilin and retards the folding of type 1 collagen and METHODS transferrin in cells (10, 11). Strong evidence for cyclophilin's Mutagenesis. Synthetic oligonucleotides were used to in- role in protein folding comes from the study of mutations in troduce insertions within the coding sequence of human the Drosophila ninaA gene, which encodes a photoreceptor- CyPB as described (22). Mutants were selected by differential specific cyclophilin. In ninaA mutant flies, two of the four hybridization of 32P-labeled mutagenic oligonucleotides and rhodopsins fail to reach the plasma membrane (12, 13). The confirmed by dideoxy sequencing with Sequenase (United mapping of various ninaA alleles places these loss-of- States Biochemical). The mutant cDNAs were cloned into function mutations at or near the putative site ofCsA binding the mammalian expression vector pECE and the in vitro and therefore the PPIase , connecting ninaA func- translation vector pSP73 (Promega). The region encoding tion to PPIase activity. These data support the view that the Asp26-Glu2O of CyPB and its mutants was cloned in-frame cyclophilins facilitate protein folding by catalyzing the into the pGEX2T bacterial expression vector (23). isomerization of specific covalent bonds. Transfections. HeLa and BHK cells were maintained in A second but potentially more important role for the Dulbecco's modified Eagle's medium (DMEM) (GIBCO) cyclophilins is as chaperone for protein trafficking and mac- supplemented with 10% fetal bovine serum (HyClone). Ten romolecular assembly. In support of chaperone function, thousand cells were plated onto each 18-mm coverslip -20 hr wild-type ninaA protein is found not only in the endoplasmic before transfection. Sixteen hours after plating, the cells were reticulum (ER) but in downstream secretory vesicles, sug- fed with fresh medium. phosphate/DNA precipi- gesting a stable association between ninaA and rhodopsin. tates were made by adding 30 ILI of 282 mM NaCl/0.78 mM Moreover, cyclophilins form stable complexes with human Na2HPO4/50 mM Hepes, pH 7.1, to 2 ,ug of supercoiled immunodeficiency virus Gag protein, an association dis- plasmid DNA in 30 gl of 200 mM CaCl2. After 20 min, 350 pl rupted by CsA (14). Further support for cyclophilin's role as of medium was added and 400 ,ld of this mixture was placed

The publication costs of this article were defrayed in part by page charge Abbreviations: CsA, cyclosporin A; PPIase, peptidyl-proline cis- payment. This article must therefore be hereby marked "advertisement" trans isomerase; CyPB, cyclophilin B; ER, endoplasmic reticulum; in accordance with 18 U.S.C. §1734 solely to indicate this fact. GST, glutathione S-. 3931 Downloaded by guest on September 23, 2021 3932 Cell Biology: Price et al. Proc. NaMl. Acad. Sci. USA 91 (1994) on the cells. Three hours later the cells were washed twice and protein composition was analyzed by SDS/polyacryl- with medium and placed in a 370C incubator (24). amide gel electrophoresis. Immunofluorescence. After 15 hr of expression, the cells PPIe Assays. PPIase activity of the fusion proteins was were fixed for 10 min in 3% formaldehyde in phosphate- determined by a protease-coupled chromogenic assay (34). buffered saline (PBS) and washed with PBS containing 0.1% Protein (0-200 nM) was assayed in a buffer containg 35mM Nonidet P-40. Myc-tagged CyPB was detected with mouse Hepes (pH 8.0), 100 ,uM tetrapeptide substrate (N-succinyl- monoclonal antibody 9E10 (31). Rabbit antiserum was made Ala-Ala-Pro-Phe p-nitroanilide), and a-chymotrypsin (Sig- against a peptide corresponding to the last 10 amino acids ma) at 250 pg/ml. Absorbance at 390 nm was sampled at (VEKPFAIAKE) of CyPB and was affinity purified on a 0.5-sec intervals in a Hewlett-Packard spectrophotometer peptide column. The rabbit polyclonal antibody (RL90) made fitted with a thermally controlled cuvette cooled to 100C. The against protein disulfide-isomerase was used to identify the CsA IC50 value was determined by a 5-min incubation with ER (26). The mouse monoclonal antibodies TC11 and 53FC3 CsA (1-1000 nM) prior to the start of the assay. (25) were used to identify the Golgi apparatus. The CD2 expression vector and the rabbit antiserum to CD2 were provided by Stephen Burakoff (Dana-Farber Cancer Insti- RESULTS tute) and Barbara Bierer (Dana-Farber Cancer Institute). CyPB Traffis lTrogh the Secretory Pathway. To track DNA was visualized with Hoechst dye 33258 (Sigma). CyPB through the secretory pathway, we introduced a 10- Pulse-Chase Experiments. BHK cells (105) were plated amino acid Myc epitope tag between the conserved cyclo- onto 60-mm plates 20 hr prior to transfection. Four hours philin domain and the 10-amino acid C-terminal domain before transfection the cells were fed with fresh medium. unique to CyPB (Fig. 1). This modified cDNA was trans- Transfections were performed essentially as described above fected into HeLa cells and the expressed protein was fol- but were scaled-up 10-fold. After 13 hr of expression, me- lowed by indirect immunofluorescence with the anti-Myc dium was replaced with DMEM lacking methionine and epitope monoclonal antibody (30, 31). Both the perinuclear cysteine (ICN) and supplemented with 2% bovine serum region and a pattern ofdots at the cell periphery were stained albumin (Sigma), penicillin/streptomycin (GIBCO), and (Fig. 1A). The diffuse perinuclear staining of CyPB colocal- L-glutamine (GIBCO). After 20 min the medium was replaced ized with an antibody directed against protein disulfide- with methionine- and cysteine-deficient medium containing isomerase (26), a soluble that resides in the ER (Fig. Tran 35S label (ICN), a mixture of 35S-labeled methionine and 1B). The presence ofCyPB in the ER is in agreement with cell cysteine, at 150 pCi/ml (1 puCi = 37 kBq). Cells were labeled for 30 min and then washed with regular medium supple- CVPA __ = eor'o a- mented with methionine (300 pg/ml) and cysteine (480 pg/ CVPB __ ml). For each time point, medium was saved and the cells a r were washed twice with PBS. After aspiration ofthe PBS, the cells were scraped from the plate in 400 A4 ofextraction buffer A Myc-tagged CyPB B PD1 (50 mM Tris, pH 7.4/100 mM NaCl/0.4% SDS/1 mM dithio- threitol/1 mM phenylmethanesulfonyl fluoride with pepsta- C)-O tin and leupeptin each at 1 pg/ml). The plate was then washed N with an additional 400 j1 of extraction buffer. The viscosity of the cell extracts was reduced by passage through a 23-gauge needle. Triton X-100 was added to 2% (vol/vol), and the extract was centrifuged at 16,000 x g at 4°C for 5 min. .E The supernatant was then transferred to a new tube contain- ing 9E10 anti-Myc antibody and protein G-coupled Sepha- C myc -tagged CyPB D DNA rose beads and incubated overnight at 4°C. The beads were then washed five times in wash buffer (50 mM Tris, pH a) 7.4/100 mM NaCl/0.1% SDS/0.5% Triton X-100/1 mM dithiothreitol/1 mM phenylmethanesulfonyl fluoride with pepstatin and leupeptin each at 1 pug/ml) and twice in 50 mM Tris (pH 7.4) and finally boiled in sample buffer (27). Glutathione S-Transerase (GST) Fusion Proteins. Esche- richia coli DH5a bacteria, transformed with the pGEX2T- CyPB plasmids, were grown to an OD590 of 0.6 and induced E endogenous CyPB F non-tagged CyPB with 200 mM isopropyl 3D-thiogalactopyranoside. After centrifugation, the pellet was suspended in lysis buffer (50 mM Tris, pH 8.0/100 mM NaCl/1 mM EDTA/1 mM dithio- threitol/1 mM phenylmethanesulfonyl fluoride with pepsta- tin and leupeptin each at 1 pg/ml) including lysozyme at 0.5 pug/ml and incubated at 4°C for 1 hr. The lysate was then sonicated. To reduce viscosity, the lysate was incubated with DNase I (5 pug/ml) and 1 mM MgCl2 for 30 min at room temperature. Finally, EDTA (10 mM) and Triton X-100 (1%) FIG. 1. Localization of CyPB in HeLa cells. CyPB has a con- were added and the extract was centrifuged 8000 x g for 10 served core domain and unique N- and C-terminal extensions. A min. Glutathione-agarose beads were incubated with the Myc-epitope tag was placed between the core domain and the supernatant for 30 min at 4°C. The beads were washed three C-terminal extension. cDNA constructs were transfected into HeLa cells. (A) Myc-tagged CyPB detected with an anti-Myc antibody. (B) times with lysis buffer. Fusion proteins were eluted from the Protein disulfide-isomerase in the same field. (C) Myc-tagged CyPB beads with 15 mM glutathione in 50 mM Tris (pH 8.0) for 1 in nonpermeabilized cells. (D) Hoechst 33258 staining of nuclei in the hr at 40C and concentrated with a microconcentrator (Ami- same field. (E and F) Endogenous CyPB and transfected nontagged con). Protein concentration was determined by Bradford CyPB, respectively, detected with an anti-peptide antibody raised assay (Bio-Rad) with bovine serum albumin as standard (28) against the C-terminal 10 amino acids of CyPB. (Bar in B = 10 ,um.) Downloaded by guest on September 23, 2021 Cell Biology: Price et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3933

fractionation and immunofluorescence studies of endoge- A no CsA R 1 iM CsA nous CyPB (29, 32). A no CsA In addition to the ER, CyPB also appeared in a pattern of -- aligned spots seen in the periphery of transfected cells (Fig. -5- . 1A). These spots did not colocalize with endosomal markers k and such as the mannose 6-phosphate receptor or transferrin w receptor (33) (data not shown). To test whether these aligned - patches were located on the plasma membrane, we per-

C:D formed immunofluorescence in the absence of detergent, -CsA CD which is usually employed to render cellular membranes m ., 0 *E C~ C~ 8 C C C permeable to antibodies. Under these conditions, the anti- *FoCCC. ' a E 0c 0 0 0 8 5 L CD 0 o0 o T Myc antibodies displayed the aligned punctate pattern of > _- 3s 5 6 7 89 10 13 14 permeabilized cells, whereas the ER appeared devoid of 15 16 17 8 signal (Fig. 1C). The labeling pattern described for CyPB in Media HeLa cells was essentially identical to that seen in other cell 31.0

lines examined, including those of simian (COS) and hamster 21 5 - (baby hamster kidney, BHK) origin (data not shown). This localization differs with previous work using anti-peptide Cell

antibodies, which showed endogenous CyPB associated with ...... 31.0- __ intracellular vesicles that contained calreticulin (29). - - -.W------11 To address the possibility that overexpression confers an 21.5-u aberrant CyPB labeling pattern, we investigated the distri- bution of endogenous CyPB by using an anti-peptide anti- body directed against the last 10 amino acids (VEKPFA- D ic Cell(-) IAKE). While the endogenous CyPB appeared less abundant 038BO 1 than tagged CyPB in transfected cells, it showed an identical perinuclear distribution which colocalized with protein di- a-3 605 CelIl+) sulfide-isomerase staining (data not shown). Additionally, 0)6 40 - Media(+) endogenous CyPB appeared in peripheral spots (Fig. 1E). 0. 20 Thus, the transfected CyPB exhibited a pattern similar to that 1 ~ ~ Media(-)J of the endogenous protein. 0 1 2 3 4 55 To control for the possibility that the epitope tag disturbs Time (hrs.) the native structure of CyPB, we transfected a nontagged FIG. 2. CsA affects CyPB distribution. (A and B) Myc-tagged CyPB construct and followed its distribution by using the CyPB was expressed in HeLa cells in either the absence (A) or the anti-peptide antibody. As with the endogenous and Myc- presence (B) of 1 ,M CsA. (C) Myc-tagged CyPB expressed in BHK tagged CyPB, the nontagged version also appeared in the ER cells was pulse-labeled with [35S]methionine/[35S]cysteine for 30 min and in foci on the cell surface (Fig. 1F). In addition, both the and, after various lengths of chase, immunoprecipitated from both wild-type and the epitope-tagged versions of CyPB were medium (upper autoradiogram) and cell lysate (lower autoradiogram) tested for PPIase activity. Both proteins were expressed in E. at 10, 40, 70, 130, 190, and 250 min after initiation of the chase. In coli as GST fusions, purified to homogeneity with glu- lanes 1 and 2, CyPB and Myc-CyPB refer to in vitro translation (IVT) tathione-agarose (23), and assayed for PPIase activity (34). products of the nontagged (22 kDa) and the Myc-tagged (28 kDa) CyPB, respectively. Duplicate pulse-chase experiments were car- levels of PPIase were detected with Wild-type activity Myc- ried out in the absence (lanes 3-10) and presence (lanes 11-18) of 1 tagged CyPB (Table 1), indicating that the Myc epitope tag AM CsA. Lanes 3 and 11 were overloaded with immunoprecipitates had no detectable effect on proper folding. from cells transfected with pECE vector alone. (D) Signals from the CsA Binding Alters the Distribution ofCyPB. CsA is a cyclic immunoprecipitates in C quantified with a Molecular Dynamics peptide that competitively inhibits the PPIase activity of Phosphorlmager. cyclophilins (35). To determine whether ligand binding af- fects CyPB trafficking through the secretory pathway, tagged The specificity of CsA's effect on CyPB distribution was CyPB was expressed in HeLa cells in the presence of 1 IM tested by cotransfection of a cDNA encoding the lymphocyte CsA. CyPB staining was virtually absent from the ER and cell surface antigen CD2 (36) in the presence of CsA. Unlike greatly reduced on the cell surface in comparison to cells the strong effect on CyPB localization, the level of CD2 on expressing CyPB without drug (Fig. 2 A and B). The most the cell surface appeared to be unaffected by 1 tuM CsA (data prominent signal remaining in the cell was a juxtanuclear not shown). Additionally, the localization of other secretory- pattern which colocalized with antibodies to mannosidase II, compartment markers, including mannosidase II and protein a Golgi complex marker (25) (data not shown). disulfide-isomerase, was unaffected by CsA. These data indicate that CsA specifically affects CyPB distribution Table 1. PPIase activity of CyPB rather than causing a general effect on proteins in the Bacterially expressed kcat/Km, CsA IC50, secretory pathway. protein g&M-1sec-1 nM CsA Promotes Mobilization and Secretion of CyPB. The CyPB 6.3 45 CsA-induced loss of CyPB from the ER and the cell surface be due to an arrest and accumulation of GST 0 ND might CyPB-CsA GST-CyPB 4.9 ND complexes in the Golgi apparatus. Alternatively, CsA binding GST-CyPB-Myc tag 4.7 40 might promote secretion of CyPB. In this case, the Golgi staining indicates a slow step in the pathway to secretion. To CyPB was produced in E. coli as either the mature protein lacking distinguish between these two models of how CsA binding a signal sequence (CyPB) or as fusions with GST (GST-CyPB and we a anal- GST-CyPB-Myc tag). Each of these proteins was tested for its affects CyPB distribution, performed pulse-chase ability to isomerize a proline residue in a tetrapeptide substrate, and ysis of CyPB in the presence and absence of CsA. BHK cells this value is expressed as catalytic efficiency, kcat/Km. The inhibition were used for this analysis because their transfection fre- of PPIase activity by CsA is presented as an IC50 concentration. quency is 10-fold greater than that of HeLa cells and the Downloaded by guest on September 23, 2021 3934 Cell Biology: Price et al. Proc. NatL. Acad. Sci. USA 91 (1994)

Signal Seq. myc tag operates to recycle ER-resident proteins back to the ER (38). To address this possibility, we cloned the 4-amino acid signal Tail / KDEL fI sequence (KDEL) specifying ER retrieval at two sites in core domain -I 0 1 CyPB (Fig. 3). When the KDEL sequence was placed at the KDEL / Tail I extreme C terminus ofCyPB, this mutant was localized to the A -(--A ER (Fig. 3A). CsA (1 pM) had no effect on the ER localization of this protein (Fig. 3B), indicating that the KDEL retrieval system was unaffected by CsA and was dominant over the CsA-induced CyPB translocation events. To control for the specificity of the KDEL insertion, we introduced a KDEL sequence at an internal site where it should be rendered nonfunctional (30). Upon transfection into cells, this mutant assumed the ER and cell surface staining seen with the wild-type and Myc-tagged CyPB proteins (Fig. 3C). Further, expression of this protein in the presence of CsA yielded a wild-type immunofluorescence pattern (Fig. 3D). This control suggests that the C-terminal KDEL CyPB mutant is indeed a substrate for the KDEL receptor. In total, these data support the notion that CsA has a specific effect on CyPB trafficking and does not disturb general protein trafficking mechanisms in the secretory path- way. FIG. 3. CsA does not affect the ER protein-retrieval system. CyPB mutants containing a KDEL sequence at the C terminus (A and DISCUSSION B) or internally (C and D) were expressed in HeLa cells in the absence (A and C) or presence (B and D) of 1 pM CsA and detected CyPB in the Secretory Pathway and In Complexes on the by anti-Myc antibody. Plasma Membrane. We have shown that human CyPB accu- mulates both in the ER and in complexes on the cell surface. effects of CsA on localization are identical. CyPB was While the localization to the ER was predicted by an ER expressed in BHK cells for 13 hr, at which time newly signal sequence in CyPB (39) and confirmed by subcellular synthesized proteins were 35S-labeled for 30 min. The me- fractionation (32), CyPB lacks domains that would explain its dium was then replaced with complete medium supplemented association with the plasma membrane. The immunofluores- with extra methionine and cysteine, and, after various times, cence data presented here indicate that these cell surface CyPB was immunoprecipitated from both medium and total complexes are often aligned or clustered on the cell surface cell lysate by use of the monoclonal antibody to the Myc and are aproperty ofboth transfected and endogenous CyPB. epitope. The abundance and apparent stability of these cell surface In the absence of CsA, we detected a single species of complexes argue in favor ofa noncatalytic role ofCyPB in the Myc-tagged CyPB (26 kDa) secreted into the medium at 40 secretory pathway, possibly as a chaperone to plasma mem- min (Fig. 2C). The mobility of the secreted species was brane proteins, in addition to its proposed function as a identical to the major product (>90%6) precipitated from PPIase in the ER. intact cells and most likely corresponded to mature CyPB Ligand-Mediated Translocation Through the Secretory lacking a signal sequence. A 28-kDa species which comi- Pathway. Ligand-receptor (or domain-domain) interactions grated with full-length in vitro translated Myc-tagged CyPB have been shown to govern the trafficking of a number of was slowly chased into the 26-kDa species in the cell (Fig. proteins in the secretory pathway, including immunoglob- 2C). It is unclear why this 28-kDa species persisted during the ulins, the influenza virus hemagglutinin protein, vesicular chase period, since signal sequences are usually cleaved stomatitis virus G protein, majorhistocompatibility complex- cotranslationally or soon after protein translocation to the encoded class I heavy chains, the KDEL receptor, and T-cell lumen of the ER (37). Part of the explanation for this receptor complexes (38, 40-47). The heavy chains of class I apparently slow cleavage event may be that a minority histocompatibility proteins remain in the ER until they bind (40%o) of the transfected cells produced very high levels of appropriate peptide ligands, which stimulate theirassociation CyPB which appeared to be deposited in the ER. This with P2-microglobulin and promote transport ofthe resulting population of cells may fail to process the signal sequences complex to the plasma membrane. Similarly, CyPB's asso- from CyPB. Regardless, over a period of 4 hr, the ratio of ciation with slower-folding plasma membrane proteins, ini- CyPB which appears in the medium increased to 18% of the tially in the ER and continuing out to the plasma membrane, total labeled CyPB. Extrapolation of these data indicates that might explain the stable localization in the ER and on the cell CyPB was released into the medium with a half-time of 418 surface. We show here that CsA causes a specific and rapid hr. translocation of CyPB from the ER into later steps of the The rate of CyPB secretion was markedly enhanced in the secretory pathway, leaving the cell surface devoid of CyPB. presence of 1 A&M CsA, as evidenced by the increased CsA might compete with newly synthesized plasma mem- accumulation in the medium and a corresponding loss from brane proteins for binding to CyPB in the ER. CsA lacks the cell (Fig. 2C). The estimated half-time for CyPB secretion domains that would anchor bound CyPB to the plasma in the presence of CsA was 4 hr. Thus, CsA resulted in a membrane and so leads to secretion of its bound CyPB. 4-fold enhancement in the rate of CyPB secretion from these Is CyPB a PPIase or a Molecular Chaperone? The obser- cells. Further, these data indicate that the accumulation of vation that CyPB traffics through the entire secretory path- CyPB-CsA complexes in the Golgi reflects a slow step in way, most likely in association with other proteins, raises their transport rather than a specific block at the Golgi questions about its function in the cell. While CyPB is an complex. active PPIase with certain small peptide substrates and CsA Does Not Affect the ER Protein-Retrieval System. The several proteins in vitro, this function in the secretory path- ability of CsA to mobilize CyPB from the ER may be due to way remains unproven. The folding of two potential sub- a general effect of this drug on the retrieval system that strates, collagen and transferrin, appears somewhat slowed Downloaded by guest on September 23, 2021 Cell Biology: Price et A Proc. Natl. Acad. Sci. USA 91 (1994) 3935 but not blocked in the presence of CsA (10, 11). Mutations in 16. Gething, M.-J. & Sambrook, J. (1992) Nature (London) 355, the Drosophila cyclophilin, ninaA lead to the accumulation of 33-45. two rhodopsin isotypes in the ER, presumably the result of 17. Spik, G., Haendler, B., Delmas, O., Mariller, C., Chamoux, M., Maes, P., Tartar, A., Montreuil, J., Stedman, K., Kocher, improper folding (13). While these data support the possibil- H. P., Keller, R., Hiestand, P. C. & Movva, N. R. (1991) J. ity that the ninaA phenotype is due to a loss of PPIase Biol. Chem. 266, 10735-10738. activity, the localization of ninaA to rhodopsin-containing 18. Caroni, P., Rothenfluf, A., McGlynn, E. & Schneider, C. (1991) vesicles in very late stages of the secretory pathway suggests J. Biol. Chem. 266, 10739-10742. additional roles for this cyclophilin (13). One of these roles 19. Bergsma, D. J., Eder, C., Gross, M., Kersten, H., Sylvester, D., Applebaum, E., Cusimano, D., Livi, G. P., McLaughlin, may be to chaperone several of the rhodopsin isotypes during M. N., Kasyan, K., Porter, T. G., Silverman, C., Dumming- theirtrafficking to the cell surface. More recently, cyclophilin ham, D., Hand, A., Prichett, W. P., Bossard, M. J., Brandt, M. A and B have been shown to specifically bind the human & Levy, M. A. (1991) J. Biol. Chem. 266, 23204-23214. immunodeficiency virus Gag protein (14). This interaction is 20. Anderson, S. K., Gallinger, S., Roder, J., Frey, J., Young, disrupted by the binding of CsA. Although the role of H. A. & Ortaldo, J. R. (1993) Proc. NatI. Acad. Sci. USA 90, cyclophilins in the infection or assembly of the virus is 542-546. 21. Cacalano, N., Chen, B.-X., Cleveland, L. & Erlanger, B. F. unclear, the Gag protein's propensity to form homooligomers (1992) Proc. NatI. Acad. Sci. USA 89, 4353-4357. might be controlled by CyPB binding in vivo (48). 22. Zoller, M. J. & Smith, M. (1984) DNA 3, 479-488. Thus, several lines of evidence argue against cyclophilins 23. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40. functioning solely through PPIase activity. They may instead 24. Heald, R. & McKeon, F. (1990) Cell 61, 579-589. function more like the heat shock proteins, by stabilizing 25. Burke, B., Griffiths, G., Reggio, H., Louvard, D. & Warren, G. partially folded intermediates, and block aggregation or pre- (1982) EMBO J. 2, 1621-1628. 26. Kaetzel, C. S., Rao, C. K. & Lam, M. E. (1987) Biochem. J. mature assembly (16, 49, 50). Resolving the function of CyPB 241, 39-47. in the secretory pathway hinges on the identification of 27. Narula, N., McMorrow, I., Plopper, G., Doherty, J., Matlin, endogenous binding proteins, such as those that anchor K., Burke, B. & Stow, J. (1992) J. Cell Biol. 117, 27-38. CyPB to the plasma membrane. 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 29. Arber, S., Krause, K. H. & Caroni, P. (1992) J. Cell Biol. 116, We thank Brian Burke for providing antibody to mannosidase II 113-126. and Charlotte Kaetzel for antibody to protein disulfide-isomerase. 30. Munro, S. & Pelham, H. R. B. (1987) Cell 48, 899-907. 31. Evan, G. I., Lewis, G. K., Ramsay, G. & Bishop, J. M. (1985) Barbara Bierer and Stephen Burakoff provided the CD2 expression Mol. Cell. Biol. 5, 3610-3616. plasmid and CD2 antibody. We thank Marten Botfield for advice in 32. Hasel, K. W., Glass, J. R., Godbout, M. & Sutcliffe, J. G. fusion protein production. We are grateful to Bill Braell, Brian (1991) Mol. Cell. Biol. 11, 3484-3491. Burke, Anjana Rao, Jun Liu, and Christine Jost for helpful comments 33. 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