©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hsp47 and Cyclophilin B Traverse the Endoplasmic Reticulum with Procollagen into Pre-Golgi Intermediate Vesicles
A ROLE FOR Hsp47 AND CYCLOPHILIN B IN THE EXPORT OF PROCOLLAGEN FROM THE ENDOPLASMIC RETICULUM (*)

(Received for publication, February 15, 1995; and in revised form, May 16, 1995)

Timothy Smith Luciano R. Ferreira Carla Hebert Kathleen Norris John J. Sauk (§)

From the Department of Pathology, School of Dentistry, University of Maryland at Baltimore, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5`-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.


INTRODUCTION

Type I procollagen is a trimeric molecule composed of two proalpha1(I) chains and one proalpha2(I) chain. Like other secretory proteins(1, 2, 3, 4) , procollagen alpha-chain synthesis begins in the cytosol with translation of a signal peptide. Subsequently, the signal sequence binds to a recognition particle that docks the complex with a membrane receptor on the endoplasmic reticulum (ER). (^1)Coincident with membrane association, the nascent protein is shifted into the membrane and traverses into the cisternal space(5, 6, 7) . Subsequently, the association of alpha-chain carboxyl propeptides provides the appropriate alignment and orientation for ensuing triple helix formation(8) .

An important feature of native triple helical structure of procollagen is that this structure can include only trans-peptide bonds. However, it has been estimated that 16% of the X-Pro bonds and 8% of the X-Hyp bonds in nascent type I collagen are in cis- rather than trans-confirmation(9) . Since, the temperature for folding kinetics of collagen is consistent with the normal Arrhenius activation energy for cis/trans-isomerization of peptide bonds, cis/trans-isomerization is considered to be a significant factor in collagen folding(10, 11, 12) . Consequently, when some of these enzymes are inhibited by the addition of cyclosporin A (CsA), the rate of in vivo folding of collagen is retarded(13) .

The cyclophilin proteins (CyPs) are a highly conserved family of PP(i)ases expressed ubiquitously in prokaryotes and eukaryotes(14, 15, 16) . These proteins act as intracellular receptors for the immunosuppressant CsA(17, 18) . The cyclophilin family, like heat shock proteins, possesses a conserved core domain flanked by variable domains at the N and C termini(15, 19) . It has been suggested that the variable domains encode subcellular targeting information. Consequently, CyPs have been classified into several isoforms; CyPA is cytosolic, while CyPB-C and the Drosophila cyclophilin, ninaA, possess ER signal sequences directed to the secretory pathway (19) . In addition to their rotamase activity (cis/trans-isomerase)(20, 21, 22, 23) , CyPs also appear to possess chaperone properties for protein trafficking and macromolecular assembly(14, 16, 24, 25, 26, 27) . For example, the Drosophila ninaA gene encodes a photoreceptor-specific CyP. Furthermore, ninaA and rhodopsin Rh 1 colocalize to secretory vesicles, suggesting the Rh 1 requires ninaA as it travels through the distal compartments of the secretory pathway(19) .

A number of ER resident proteins, designated as molecular chaperones, have been associated with polysome-associated pro-alpha1(I) chains(28, 29) . These have included the GRP78/Bip, GRP94, protein disulfide isomerase (PDI), and Hsp47, which appear to function in a series of coupled or successive reactions during procollagen production and assembly(30) . While investigating the relationship between various molecular chaperones and evolving procollagen chains associated with dense polysomes, a 20.6-kDa protein was observed to be closely associated with procollagen, Hsp47, PDI, GRP78, and GRP94. Here, we report this 20.6-kDa protein to be a CyPB that associates early in the translation/translocation of procollagen and may function in consort with other ER resident proteins to prevent aggregation of nascent chains, mediate proline isomerization(3) , and possibly improve the catalytic effect of protein disulfide isomerase(31) . Also, CyPB and Hsp47 were localized to the ER and to pre-Golgi intermediate vesicles associated with procollagen, suggesting that Hsp47 and CyPB have a role in procollagen sorting and transport.


MATERIALS AND METHODS

Cell Culture and Metabolic Labeling

Mouse 3T6 cells obtained from the American Type Tissue Collection were used in all experiments. The cells were grown and maintained in plastic flasks using Dulbecco's modified Eagle's medium, 1.16 g/liter glutamine, 10% fetal bovine serum, 10 µg/ml ascorbate, 100 units of penicillin, and 100 µg/ml streptomycin at 37 °C. In those instances when it was necessary to label proteins, the medium was removed and replaced with fresh methionine-free Dulbecco's modified Eagle's medium containing [S]methionine (100 µCi/ml, DuPont NEN) for varying periods (see figure legends).

Isolation of Intact Polysomes and Nascent Procollagen

Dense polysomes were prepared after a modified protocol of Kirk et al.(32) . In essence, Mouse 3T6 cells were grown to near confluence as described above, and protein synthesis was blocked by the addition of cycloheximide (100 µg/ml) for 10 min. In some instances, cross-linking with dithiobis(succinimidyl propionate) was used to determine the near neighbors of polysome-associated proteins(28) . The cells were suspended in buffer A (0.2 M Tris-HCl, pH 7.4, 0.24 M KCl, 0.0075 M MgCl(2), 0.1 mg/ml cycloheximide, 0.2 mg/ml heparin, 2 mM dithiothreitol, 0.05% sodium deoxycholate, 0.16 mg/ml phenylmethylsulfonyl fluoride, and 0.78 mg/ml benzamidine), and Triton X-100 was then added to a final concentration of 2%. The cells were homogenized and centrifuged at 10,000 g for 30 min to remove nuclei and cellular debris. The resulting supernatant was collected and the volume adjusted to 5 ml with buffer A. The supernatant (2.5 ml) was then layered on top of 1 ml of 1 M sucrose layered over 1.5 ml of 2 M sucrose. The samples were centrifuged for 12 h in a Beckmann SW 55 Ti rotor at 100,000 g. Polysomes were collected, washed with water, suspended in Q-Sepharose buffer (0.02 M Tris-HCl, pH 7.4, 0.24 M KCl, 0.0075 M MgCl(2)), and applied to a 3-ml Q-Sepharose Fast Flow column (Pharmacia Biotech Inc.) as described by Bergman and Kuehl (33) and modified by Kirk et al.(32) . The resulting flow-through fraction contained tRNA-free nascent chains. The retained tRNA-bound material was eluted with 1.0 M NaCl.

N-terminal Sequencing

Proteins separated by SDS-PAGE were electroblotted onto Problot membranes at 50 mA for 3 h. The Problot was then soaked in blotting buffer (25 mM Tris, 192 mM glycine, 20% methanol). The Problot was stained with Coomassie Blue G for 30 s and then destained using acetic acid/methanol/water (1:1:18, v/v) for 12 h. The membranes were dried, and the PP(i) band was cut for N-terminal sequencing.

Reverse Transcriptase Polymerase Chain Reaction

Total RNA was isolated from approx10^8 3T6 mouse cells using a Total RNA Isolation System (Promega). The first strand cDNA synthesis reaction was catalyzed by superscript II RNase H-reverse transcriptase (RT) (Life Technologies, Inc.) with either of two 5`-end-specific primers and one 3`-end-specific primer for mouse CyPB(34) . The resulting products were analyzed by SDS-PAGE using beta-actin primers and product as a control(35) . The polymerase chain reaction-amplified fragments were ligated into the Invitrogen TA cloning system and subsequently sequenced to verify the polymerase chain reaction product. Sequencing was achieved using the Sequenase kit 2.0 (U. S. Biochemical Corp.).

PAGE and Immunoblotting

Samples for gel electrophoresis were suspended in Laemmli SDS-PAGE buffer and boiled for 5 min before loading onto 4-20% polyacrylamide gradient slab gels after the method of Laemmli(36) . The gels were fixed, dried, and autoradiographed using the method of Bonner and Laskey(37) .

For Western blots, proteins run on SDS-PAGE were immediately electrotransferred to nitrocellulose paper. The paper was blocked with 10% non-fat dry milk in 10 mM Tris-HCl, pH 7.4, 0.9 M NaCl (TBS) for 1 h and then in TBS/non-fat dry milk with 2% normal goat serum (Life Technologies, Inc.). Antiserum or preimmune serum was diluted 1:2000 in the same buffer and incubated with gentle shaking overnight. The nitrocellulose was then rinsed three times for 5 min in TBS/Tween. The secondary antibody, affinity-purified biotinylated goat-anti-rabbit IgG (Fc) (Kirkegaard and Perry Labs, Gaithersburg, MD) was diluted to 0.9 µg/ml and incubated with the paper for 2 h. Washing between steps was performed three times for 30 min with 50 mM Tris-HCl, 0.9 M NaCl, 0.05% Tween, pH 7.4. The blot was visualized using ECL Western blot protocol (Amersham Corp.). Hsp47 rabbit polyclonal antibodies were prepared against a 22-mer peptide corresponding to the N-terminal sequence of mouse Hsp47 that was conjugated to Keyhole limpet hemocyanin and was cross-reactive with mouse 3T6 Hsp47(38) . Polyclonal rabbit anti-mouse alpha1(I) procollagen antibodies were made from acid-soluble procollagen derived from lathyritic mouse skin(28) . Polyclonal rabbit antibodies were made against a peptide corresponding to the C-terminal decapeptide (VEKPFAIAKE) of CyPB (34) and affinity purified on a peptide column. The antibodies were further characterized by Western blot to CyPB that was expressed in Escherichia coli containing the CyPB expression plasmid(34) . Notably, these antibodies failed to show any reactivity to purified CyPA (Sigma) in Western blots.

Immunoprecipitation and Analysis by Gel Electrophoresis

Cells were freed from the support medium with trypsin (0.025% EDTA (0.02%) and treated with bacterial collagenase (0.01%) and 10% fetal calf serum for 3 min to block trypsin activity and remove extracellular collagen. The samples were suspended in a equal volume of 2 immunoprecipitation buffer (0.2 M Tris-HCl, 0.3 M NaCl, 2% Triton X-100, 2% deoxycholate, 0.2% SDS, pH 7.2 containing apyrase (Sigma) to deplete ATP). The samples were centrifuged for 5 min at 10,000 g in an Eppendorf centrifuge, and a 50-µl sample of the radiolabeled supernatant was added to a mixture of protein A-Sepharose and antibody in PBS-azide. The samples were then incubated at 4 °C with constant shaking and then centrifuged at 10,000 g for 10 min. The resulting immunoprecipitates were then washed twice with PBS-azide. The final pellets were suspended in 2 gel electrophoresis sample buffer, heated for 10 min at 90 °C, and then centrifuged to remove protein A-Sepharose. Samples of the supernatants were counted in a scintillation counter, and another sample was analyzed by PAGE and autoradiography as described above.

Modulation of ER to Golgi Transport

To limit procollagen transport to pre-Golgi intermediate vesicles, cells were permeabilized with digitonin (39) and incubated in the presence of 25 µM GTPS for various periods up to 60 min (40) . To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan (17 µm)(40) . Also, in some experiments cells were treated with calphostin C, a specific inhibitor of the highly conserved cysteine-rich C(6)H(2) motif present in the regulatory domain of protein kinase C(41) . For these studies, 120 nm of calphostin C was added from a stock solution in dimethyl sulfoxide. To ensure that inhibition by calphostin was specific, parallel control samples were run in the dark since this inhibitor behaves as a caged substrate when activated by exposure to light(41) . In some experiments, cells were treated with 100 µg/ml cycloheximide to inhibit further protein synthesis.

Indirect Immunofluorescence

Fixed cells were blocked with 5% swine serum in PBS, and the Cell membranes were permeabilized with 0.1% saponin before incubation with antibody(39) . Before the addition of the second primary reagent to detect Golgi marker proteins (alpha-1,2-mannosidase II), cells were permeabilized with 0.1% saponin for 20 min in PBS/swine. Cells were subsequently washed and exposed to the primary antibody. Rabbit primary antibodies were detected with either a fluorescein isothiocyanate goat anti-rabbit IgG or Texas Red goat anti-rabbit IgG (Molecular Probes). Coverslips were mounted in Moviol (Calbiochem-Behring Corp.) and viewed in Axiovert microscope (Carl, Zeiss, Oberochen, Germany).

PPase Activity

PP(i) activity was determined in a standard coupled chymotrypsin assay utilizing N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide(42) .


RESULTS AND DISCUSSION

To study procollagen processing, 3T6 mouse fibroblasts were labeled with [S]methionine, and dense procollagen polysomes were prepared after the method of Kirk et al.(32) . The resulting polysomes were then fractionated using Q-Sepharose Fast Flow chromatography to yield two pools of procollagen. One pool consisted of elongating procollagen bound to peptidyl-tRNA that was initially retained (RT) and eluted with salt. The flow-through fraction consisted of recently completed nascent chains that were disrupted during column fractionation. The eluted RT fraction was then immunoprecipitated with anti-collagen I antibodies. Hsp47 was noted to coimmunoprecipitate with nascent procollagen I alpha chains. In addition to Hsp47, GRP78, and GRP94, an additional 20.6-kDa protein was recovered in immunoprecipitates associated with peptidyl tRNAs (Fig. 1). Measurement of the PP(i)ase activity of the dense polysome preparation, Q-Sepharose fractions, and immunoprecipitates revealed an enrichment in PP(i)ase activity (Table 1).


Figure 1: Q-Sepharose fractions of [S]methionine-labeled dense polysomes derived from 3T6 cells. The toppanel depicts the fractionation of dense polysomes by Q-Sepharose chromatography. LaneA depicts the flow-through fraction consisting of recently completed nascent chains disrupted during column fractionation. LaneB depicts the elongating chains bound to peptidyl tRNA that was initially retained (RT) and eluted with high salt. LaneC represents the RT fraction that was digested with bacterial collagenase subsequent to precipitation. The bottompanel depicts an immunoprecipitate of a dithiobis(succinimidyl propionate) cross-linked RT fraction obtained with anti-collagen I antibodies. The various proteins labeled in the figure were identified by susceptibility to bacterial collagenase and Western blot analysis. The proteins identified were as follows: lane1, Hsp47; lane2, PDI; lane3, CyPB; lane 4, GRP94; lane5, GRP78.





Previous studies had also shown that microsomes may possess a high PP(i) activity that is inhibited by CsA. Furthermore, this activity was attributed to a protein belonging to the CyPB family(43) . To ascertain if the 20.6-kDa protein precipitating with procollagen nascent chains was a member of the CyPB family, the RT fraction was separated by PAGE electroblotted onto Problot membranes, and the 20.6-kDa band was cut out of the membrane for N-ternimal sequencing. The resulting sequence NDKKKGPKVTVKVYFDLQIG was identical to that previously reported for mouse CyPB (34) being rich in lysine and thus distinct from mitochondrial cyclophilins that possess a serine-rich N-terminal extension(44) .

To further characterize this protein, we sought to determine if this protein possessed an N-terminal signal sequence that directs the protein to the ER and a C-terminal decapeptide extension distinct from cytosolic and mitochondrial forms of cyclophilin(43) . To address these questions, total RNA was isolated from approx10^8 3T6 mouse cells using a total RNA isolation system (Promega). The first strand cDNA synthesis reaction was then catalyzed by SuperScript II RNase H-RT (Life Technologies, Inc.). Next, the protein-coding region was amplified by the polymerase chain reaction (45) with either of two 5`-end-specific primers and one 3`-end-specific primer (see ``Materials and Methods''). The polymerase chain reaction-amplified fragments were ligated into the Invitrogen TA cloning system and subsequently sequenced. These results verified that the amplified products from mouse 3T6 cells were identical to CHP2 (CyPB)(34) . As such, this sequence contained 25 amino acids (MKVLFAAALIVGSVVFLLLPGPSVA) that resembled a signal sequence and a predicted cleavage site between Ala-25 and Asn-26, in which the resultant protein would result in a 20.6-kDa final product. In addition, the protein concluded with a C-terminal VEKPFAIAKE, distinct from cytosolic and mitochondrial cyclophilins(43, 44) .

Affinity-purified rabbit antibodies prepared against a synthetic peptide corresponding to the C-terminal decapeptide of CyPB in Western blots showed reactivity against the 20.6-kDa protein in immunoprecipitates obtained with anti-collagen or anti-Hsp47 antibodies (Fig. 1). Also, when the dense polysome Q-Sepharose-retained fraction was immunoprecipitated with anti-CyPB antibodies, a number of proteins were noted to coimmunoprecipitate with CyPB. Some of these proteins were identified by susceptibility to digestion by bacterial collagenase and/or Western blot analysis as procollagen, PDI, Hsp47, and the translocon component Sec61p (Fig. 2).


Figure 2: Immunoprecipitate obtained with anti-CyPB antibodies of dithiobis(succinimidyl propionate) cross-linked dense polysomes derived from 3T6 cells. Dense polysomes prepared and fractionated as described in Fig. 1were immunoprecipitated with anti-CyPB antibodies. LaneA represents the immunoprecipitate digested with bacterial collagenase (28) prior to SDS-PAGE. LaneB depicts the immunoprecipitate obtained with anti-CyPB antibodies. The proteins labeled in the figure were confirmed by Western blot. The proteins identified were laneC, Sec61p; laneD, Hsp47; laneE, CyPB; laneF, GRP78; laneG, GRP94.



To determine whether CyPB was retained within the ER as previously suggested (45) or implied further in the secretory pathway, cells were pulse labeled with [S]methionine chased in cold medium in excess of the labeled amino acid and immunoprecipitated with various antibodies. These data revealed that Hsp47 and CyPB were both closely associated with procollagen during the first 10 min of the chase period. However, subsequently both proteins progressively dissociated from procollagen and were unassociated with secreted procollagen in the medium (Fig. 3).


Figure 3: Kinetics of total cellular procollagen, Hsp47-bound procollagen, and CyPB-bound procollagen. Pulse-chase experiments were performed by labeling cells with 100 µCi/ml [S]methionine for 20 min; the cells were then chased in medium containing an excess of unlabeled methionine. Cells were harvested and lysed, and samples were immunoprecipitated with anti-procollagen antibodies (total procollagen), anti-Hsp47 antibodies (Hsp47-procollagen), and CyPB antibodies (CyPB-procollagen) and separated by SDS-PAGE and autoradiograms prepared (see ``Materials and Methods''). The bands representing procollagen I alpha-chains were scanned using a densitometer. The value of the relative density before chasing was designated as 1 unit. The mean values and the standard deviations from at least three independent experiments are plotted.



To limit procollagen secretion to certain compartments within the secretory pathway, cells were treated with agents that inhibited diacylglycerol/phorbol ester-binding proteins (41) or multiple GTP-binding proteins including heterotrimeric G protein(s)(40) . Calphostin C, a specific inhibitor of the highly conserved cysteine-rich C(6)H(2) motif present in the regulatory domain of protein kinase C, is known to be a potent inhibitor of vesicular budding from the ER (41) and was utilized to retain procollagen in the ER without directly modifying the procollagen post-translational modification. Export from the ER was also inhibited by mastoparan, a peptide that mimics G protein-binding regions of seven transmembrane-spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors(40) . In both instances, immunoprecipitation of the procollagen revealed that Hsp47 and CyPB remained bound to procollagen compared to untreated cells ( Fig. 4and Fig. 5). These results are consistent with the hypothesis that multiple GTP-binding proteins (40) and a novel protein containing a C(6)H(2) motif serve as a link in a signaling pathway regulating vesicle budding from the ER(41) . To ensure that both inhibitors were effective, the media were collected, and proteins were precipitated and subjected to SDS-PAGE. In both instances, no radiolabeled procollagen was detected in the medium. In addition, cells were also monitored by indirect immunofluorescence to verify that procollagen was retained within the ER. These studies confirmed that cells treated with either calphostin C and mastoparan retained procollagen, Hsp47, and CyPB to the ER region (Fig. 6).


Figure 4: Comparison of kinetics of Hsp47 bound procollagen in control, mastoparan, calphostin C, and GTPS-treated 3T6 cells. Pulse-chase experiments were performed as described above, and the samples were immunoprecipitated with anti-procollagen antibodies separated by SDS-PAGE and autoradiograms prepared (see ``Materials and Methods''). The bands representing Hsp47 were scanned using a densitometer. The value of the relative density before chasing was designated as 1 unit. The mean values and the standard deviations from at least three independent experiments are plotted.




Figure 5: Comparison of kinetics of CyPB-bound procollagen in control, mastoparan, calphostin C, and GTPS-treated 3T6 cells. Pulse-chase experiments were performed as described above, and samples were immunoprecipitated with anti-procollagen antibodies separated by SDS-PAGE and autoradiograms prepared (see ``Materials and Methods''). The bands representing CyPB were scanned using a densitometer. The value of the relative density before chasing was designated as 1 unit. The mean values and the standard deviations from at least three independent experiments are plotted.




Figure 6: A, distribution of Hsp47, CyPB, and procollagen I in 3T6 cells after modulation of ER to Golgi transport. Cells were incubated for 40 min in the absence or presence of mastoparan, calphostin C, or GTPS. The distribution of proteins was monitored using the cis/medial Golgi marker alpha-1,2-mannosidase II. Columna, row1 represents control cells labeled with anti-Hsp47 antibodies. Columnb, row1 depicts Hsp47 limited to the ER in mastoparan-treated cells; similar results were obtained with calphostin C. Columnc, row1 reveals the distribution of Hsp47 in ER and punctate vesicles in cells treated with GTPS. Columna, row2 depicts the distribution of CyPB in control cells. Columnb, row2 depicts CyPB in the ER of cells treated with calphostin C; similar results were obtained with mastoparan. Columnc, row3 demonstrates CyPB both in the ER and in punctate pre-Golgi vesicles following GTPS treatment. Columna, row3 depicts procollagen in control cells. Columnb, row3 represents procollagen retained in the ER following treatment with mastoparan; identical results were obtained with the use of calphostin C. Columnc, row3 represents the distribution of procollagen I in punctate pre-Golgi vesicles following 40 min of treatment with GTPS. B, distribution of Hsp47 and Golgi markers in 3T6 cells. Following treatment of cells with GTPS, Hsp47 was localized to ER and punctate vesicles (panel a). In control cells (panelb), Hsp47 was localized primarily to the ER with only a few vesicles apparent. Arrows indicate staining of Golgi with alpha-1,2-mannosidase II.



In previous immunocytochemical studies, we noted that Hsp47 was colocalized with procollagen in what appeared to be pre-Golgi vesicular structures during tooth development(46) . To verify this relationship, cells were treated with GTPS, a nonhydrolyzable analog of GTP that blocks uncoating of both ER and Golgi transport-derived vesicles(47, 48) . These studies revealed accumulation of transported procollagen and Hsp47 in pre-Golgi intermediate vesicles distributed throughout the cytoplasm of the cell as well as the ER (Fig. 6). Immunoprecipitation of procollagen in GTPS-treated cells revealed that labeled Hsp47 and CyPB coprecipitated with labeled procollagen ( Fig. 4and Fig. 5), in that CyPB trafficking through the secretory pathway can be altered by CsA (27) . Mouse 3T6 cells were treated with 1 µM CsA, labeled, and chased as described above for 60 min (Fig. 7). These studies revealed that CsA treatment diminished the levels of CyPB bound to procollagen and delayed the rate of Hsp47 release from procollagen and the rate of procollagen secretion. These data sustain previous studies that revealed that CyPB traverses the secretory pathway rather than acting solely as a proline isomerase functioning within the ER. Albeit CyPB has been shown to act as a PP(i)ase for procollagen(13) , these results suggest that the release of Hsp47 from procollagen is driven by helix formation, thereby providing one mechanism by which CyPB plays a role in procollagen export and secretion.


Figure 7: Kinetics of total cellular procollagen, Hsp47-bound procollagen, and CyPB-bound procollagen following treatment of cells with cyclosporin A. Cells were treated with 1 µM cyclosporin A for 1 h. Pulse-chase experiments were then performed by labeling cells with 100 µCi/ml [S]methionine for 20 min; the cells were then chased in medium containing an excess of unlabeled methionine. Cells were harvested and lysed, and samples were immunoprecipitated with anti-procollagen antibodies (total procollagen), anti-Hsp47 antibodies (Hsp47-procollagen), and CyPB antibodies (CyPB-procollagen) and separated by SDS-PAGE and autoradiograms prepared (see ``Materials and Methods''). The bands representing procollagen I alpha-chains were scanned using a densitometer. The value of the relative density before chasing was designated as 1 unit. The mean values and the standard deviations from at least three independent experiments are plotted.



Although previous studies have demonstrated that Hsp47 and other molecular chaperones remain bound to altered procollagen in alpha,alpha-dipyridyl-treated cells and procollagen in some forms of osteogenesis imperfecta(29, 49, 50) . The studies reported here indicate that Hsp47 association with procollagen is also compartment dependent. In that Hsp47 has been shown not to undergo Golgi processing of its N-linked oligosaccharides, we anticipated that Hsp47 was involved early in procollagen assembly(28) . However, the association of Hsp47 with procollagen in the intermediate compartment residing between the ER and Golgi suggests a further role for Hsp47 in mediating procollagen export from the ER. This raises questions as to the role of Hsp47 during transport from the ER to the Golgi.

Currently, evidence is accruing that suggests that proteins are sorted and concentrated during export from the ER. One paradigm that has been advocated presumes that the protein to be transported is insensitive and relies on the passive movement of exported proteins into designated regions of the ER specialized in export(51) . However, to ensure net concentration of the protein, it appears that a mechanism must exist to prevent backflow. To account for this, two additional models that hinge on the interaction of the transported protein with transport machinery have been proposed(51) . One of the schemes is analogous to the process of receptor-mediated endocytosis to concentrate-transported protein (52) . The other suggests that signals also participate in the active recruitment of coat components, leading to the formation of nascent budding sites(40, 51, 53) . In both instances, release from a chaperone-mediated retention system is suggested as the first step in a more regulated pathway involving multiple transport components that initiate, facilitate, or enhance the efficiency of transport(51) .

We suggest that there is an additional alternative for procollagen. In this system, Hsp47 is seen to possess both chaperone and anti-chaperone properties similar to that recently demonstrated for PDI(54) . Thus, during translation the high levels of Hsp47 function as a chaperones and limit protein aggregation and facilitate chain registration and/or posttranslational modification of procollagen(28) . Later, as procollagen concentrations increase and are released from GRP78 and GRP94, Hsp47 and/or in consort with CyPB and PDI act as anti-chaperones and provide the basis for the concentration of procollagen destined for export. However, at present it is still not clear whether the Hsp47 and CyPB that escorts procollagen into pre-Golgi intermediate vesicles initiate and/or coordinate the dynamics of coat assembly on the lipid bilayer. It will be important now to examine the transport of procollagen after alteration of Hsp47 and CyPB to define the general need for sorting and concentration and the nature of plausible signals involved in conveyance control from the ER.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DE08648 and AR41572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, School of Dentistry, University of Maryland, 666 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-7936; Fax: 410-706-0193; jjs001{at}dental3.ab.umd.edu.

^1
The abbreviations used are: ER, endoplasmic reticulum; CyP, cyclophilin; CyPB, cyclophilin B; PDI, protein disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcriptase; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; CsA, cyclosporin A; GTPS, guanosine 5`-3-O-(thio)triphosphate.


ACKNOWLEDGEMENTS

We thank JoAnn Walker for help in preparing the manuscript.


REFERENCES

  1. Walter, P., and Blobel, G.(1981)J. Cell Biol.91,557-561 [Abstract]
  2. Gilmore, R., Walter, P., and Blobel, G.(1982)J. Cell Biol.95,463-469 [Abstract]
  3. Gilmore, R., Walter, P., and Blobel, G.(1982)J. Cell Biol.95,470-477 [Abstract]
  4. Meyer, D. I., Krause, E., and Dobberstein, B.(1982)Nature297,647-650 [Medline] [Order article via Infotrieve]
  5. Simon, S. M., and Blobel, G.(1991)Cell65,371-380 [Medline] [Order article via Infotrieve]
  6. High, S., Grlich, D., Wiedmann, M., Rapoport, T. A., and Dobberstein, B.(1991)J. Cell Biol.113,35-44 [Abstract]
  7. Kellaris, K. V., Bowen, S., and Gilmore, R.(1991)J. Cell Biol. 114,21-33 [Abstract]
  8. Mayne, R., and Burgeson, R. E. (1987) Structure and Function of Collagen Types, Academic Press, Orlando, FL
  9. Sarkar, S. K., Young, P. E., Sullivan, C. E., and Torchia, D. A.(1984)Proc. Natl. Acad. Sci. U. S. A.81,4800-4803 [Abstract]
  10. Bachinger, H. P., Morris, N. P., and Davis. J. M.(1993)Am. J. Med. Genet.45,152-162 [Medline] [Order article via Infotrieve]
  11. Fischer, G., Wittmann-Leibold, B., Lang, K., Kiefhaber, T., and Schmid, F. X. (1989)Nature337,476-478 [CrossRef][Medline] [Order article via Infotrieve]
  12. Siekierka, J. J., Staruch, M. J., Hung, S. H. Y., and Sigal, N. H.(1989) J. Immunol.143,1580-1583 [Abstract/Free Full Text]
  13. Steinmann, B., Bruckner, P., and Superti-Furga, A.(1991)J. Biol. Chem. 266,1299-1303 [Abstract/Free Full Text]
  14. Hohman, R. J., and Hultsch, T.(1990)New Biol.2,663-672 [Medline] [Order article via Infotrieve]
  15. Stamnes, M. A., and Zuker, C. S.(1990)Curr. Opin. Cell Biol. 2,1104-1107 [Medline] [Order article via Infotrieve]
  16. Schreiber, S. L. (1991)Science251,283-287 [Medline] [Order article via Infotrieve]
  17. Quesniaux, V. F. J., Schreier, M. H., Wenger, R. M., Hiestand, P. C., Harding, M. W., and Van Regenmortel, M. H. V.(1987)Eur. J. Immunol. 17,1359-1365 [Medline] [Order article via Infotrieve]
  18. Borel, J. F. (1976)Immunology31,631-641 [Medline] [Order article via Infotrieve]
  19. Colley, N. J., Baker, E. K., Stamnes, M. A., and Zuker, C. S.(1991)Cell 67,255-263 [Medline] [Order article via Infotrieve]
  20. Lang, K., Schmidt, F. X., and Fischer, G.(1987)Nature329,268-270 [CrossRef][Medline] [Order article via Infotrieve]
  21. Bachinger, H. P. (1987)J. Biol. Chem.262,17144-17148 [Abstract/Free Full Text]
  22. Davis, J. M., Boswell, B. A., and Bachinger, H. P.(1989)J. Biol. Chem. 264,8956-8962 [Abstract/Free Full Text]
  23. Kiefhaber, T., Quaas, R., Hahn, U., and Schmidt, F. X.(1990)Biochemistry 29,3053-3061 [Medline] [Order article via Infotrieve]
  24. Caroni, P., Rothenfluh, A., McGlynn, E., and Schneider, C.(1991)J. Biol. Chem.266,10739-10742 [Abstract/Free Full Text]
  25. Spike, G., Haendler, B., Delmas, O., Mariller, C., Chamoux, M., Maes, P., Tarter, A., Montreuli, J., Stedman, K., Kocher, H. P., Keller, R., Hiestand, P. C., and Movva, N. R.(1991)J. Biol. Chem.266,10735-10738 [Abstract/Free Full Text]
  26. Luban, J., Bossolt, K. L., Franke, E. K., Kalpana, G. V., and Goff, S. P.(1993) Cell73,1067-1078 [Medline] [Order article via Infotrieve]
  27. Price, E. R., Jin, M., Lim, D., Pati, S., Walsh, C. T., and McKeon, F. D.(1994) Proc. Natl. Acad. Sci. U. S. A.91,3931-3935 [Abstract]
  28. Sauk, J. J., Smith, T., Norris, K., and Ferreira, L.(1994)J. Biol. Chem. 269,3941-3946 [Abstract/Free Full Text]
  29. Nakai, A., Satoh, M., Hirayoshi, K., and Nagata, K.(1992)J. Cell Biol. 117,903-914 [Abstract]
  30. Ferreira, L. R., Norris, K., Smith, T., Hebert, C., and Sauk, J. J.(1994)J. Cell. Biochem.56,518-526 [Medline] [Order article via Infotrieve]
  31. Schoenbrunner, E. R., and Schmidt, F. X.(1992)Proc. Natl. Acad. Sci. U. S. A.89,4510-4513 [Abstract]
  32. Kirk, T. Z., Evans, J. S., and Veis, A.(1987)J. Biol. Chem.262,5540-5545 [Abstract/Free Full Text]
  33. Bergman, L. W., and Kuehl, W. M.(1977)Biochemistry16,4490-4497 [Medline] [Order article via Infotrieve]
  34. Hasel. K. W., Glass, J. R., Godbout, M., and Sutcliffe, J. G.(1991)Mol. Cell. Biol.11,3484-3491 [Medline] [Order article via Infotrieve]
  35. Yokai, H., Natsuyama, S., Iwai, M., Noda, Y., Mori, T., Mori, K., Fujita, K., Nakayama, H., and Fujita, J.(1993)Biochem. Biophys. Res. Commun. 195,769-775 [CrossRef][Medline] [Order article via Infotrieve]
  36. Laemmli, U. K. (1970)Nature227,680-685 [Medline] [Order article via Infotrieve]
  37. Bonner, W. M., and Laskey, R. A.(1974)Eur. J. Biochem.46,83-88 [Medline] [Order article via Infotrieve]
  38. Sauk, J. J., Van Kampen, C. L., Norris, K., Foster, R. A., and Somerman, M. J.(1990) Biochem. Biophys. Res. Commun.172,135-142 [Medline] [Order article via Infotrieve]
  39. Plunter, H., Davidson, H. W., Saraste, J., and Balch, W. E.(1992)J. Cell Biol.119,1097-1116 [Abstract]
  40. Schwaninger, R., Plunter, H., Bokoch, G. M., and Balch, W. E.(1992)J. Cell Biol.119,1077-1096 [Abstract]
  41. Fabbri, M., Bannykh, S., and Balch, W.(1994)J. Biol. Chem.269,26848-26857 [Abstract/Free Full Text]
  42. Fischer, G., Bang, H., and Mecl, C.(1984)Biomed. Bichim. Acta43,1101-1111
  43. Bose, S., Mucke, M., and Freedman, R. B.(1994)Biochem. J.300,871-875 [Medline] [Order article via Infotrieve]
  44. Connern, C. P., and Halestrap, A. P.(1992)Biochem. J.284,381-385 [Medline] [Order article via Infotrieve]
  45. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A.(1988)Science239,487-491 [Medline] [Order article via Infotrieve]
  46. Shroff, B., Smith, T., Norris, K., Pileggi, R., and Sauk, J. J.(1993)Conn. Tissue Res.29,273-286 [Medline] [Order article via Infotrieve]
  47. Melancon, P., Glick, B. S., Malhotra, V., Weidman, P. J., Serafini, T., Gleason, M. L., Orci, L., and Rothman, J. E.(1987)Cell.51,1053-1062 [Medline] [Order article via Infotrieve]
  48. Beckers, C. J. M., and Balch, W. E.(1989)J. Cell Biol.108,1245-1256 [Abstract]
  49. Chessler, S. D., and Beyers, P. H.(1992)J. Biol. Chem.267,7751-7757 [Abstract/Free Full Text]
  50. Chessler, S. D., and Beyers, P. H.(1993)J. Biol. Chem.268,18226-18233 [Abstract/Free Full Text]
  51. Balch, W. E., McCaffery, J. M., Plunter, H., and Farquhar, M. G.(1994)Cell 76,841-852 [Medline] [Order article via Infotrieve]
  52. Schmid, S. L. (1993)Trends Cell Biol.3,145-148 [Medline] [Order article via Infotrieve]
  53. Balch, W. E. (1992)Curr. Biol.2,157-160 [Medline] [Order article via Infotrieve]
  54. Puig, A., and Gilbert, H. F.(1994)J. Biol. Chem.269,7764-7771 [Abstract/Free Full Text]

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