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-proline cis-trans isomerase/protein 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 protein folding studies. pathway via an endoplasmic reticulum 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 proteins, the cyclophilin cyclosporin A specifically mobilizes cyclophilin B from the family of proteins contains a conserved core domain flanked endoplasmic reticulum, and promotes the secretion 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 isomerases (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 calcineurin, a phosphatase 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 active site, 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. Calcium 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-transferase. 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 enzyme 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).