To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo andin vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed. To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo andin vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed. Current models of protein translocation across the mammalian endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; RM, rough microsomes; WT, wild type, FA, folding mutant; BiP, immunoglobulin heavy chain-binding protein; pPL, preprolactin; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NEM, N-ethylmaleimide. 1The abbreviations used are: ER, endoplasmic reticulum; RM, rough microsomes; WT, wild type, FA, folding mutant; BiP, immunoglobulin heavy chain-binding protein; pPL, preprolactin; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; NEM, N-ethylmaleimide. depict translocation as a process in which vectorial transport accompanies formation of a tight junctional complex between the ribosome and the protein conducting channel with the free energy for translocation provided by passive diffusion (1Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (708) Google Scholar, 2Rapoport T.A. Jungnickel B. Katay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (491) Google Scholar). In alternative models, vectorial transport may also be driven through interaction of the nascent chain with lumenal molecular chaperones (3Neupert W. Hartl F.-U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 4Simon S.M. Peskin C.S. Oster G.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3770-3774Crossref PubMed Scopus (361) Google Scholar, 5Nicchitta C.V. Blobel G. Cell. 1993; 73: 989-998Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 6Sanders S.L. Whitfield K.M. Vogel J.P. Rose M.D. Schekman R.W. Cell. 1992; 69: 353-365Abstract Full Text PDF PubMed Scopus (273) Google Scholar), as well as structural modifications of the nascent chain, i.e. protein folding, disulfide bond formation and, in many cases, addition ofN-linked oligosaccharides, that occur coincident with translocation (7Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (773) Google Scholar, 8Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1566) Google Scholar, 9Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (557) Google Scholar, 10Rothman J.E. Cell. 1989; 59: 591-601Abstract Full Text PDF PubMed Scopus (626) Google Scholar, 11Gething M.-J. Sambrook J. Nature. 1992; 355: 33-47Crossref PubMed Scopus (3565) Google Scholar). In the latter model, interactions between the nascent chain and lumenal molecular chaperones are thought to prevent retrograde transport through the translocation pore and thus bias movement of the nascent chain into the lumenal compartment (3Neupert W. Hartl F.-U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 4Simon S.M. Peskin C.S. Oster G.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3770-3774Crossref PubMed Scopus (361) Google Scholar, 5Nicchitta C.V. Blobel G. Cell. 1993; 73: 989-998Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 6Sanders S.L. Whitfield K.M. Vogel J.P. Rose M.D. Schekman R.W. Cell. 1992; 69: 353-365Abstract Full Text PDF PubMed Scopus (273) Google Scholar,12Nicchitta C.V. Semin. Cell. Dev. Biol. 1996; 7: 497-503Crossref Scopus (3) Google Scholar). Recent reconstitution experiments have identified the minimum subset of ER proteins necessary for in vitro protein translocation in the mammalian ER (13Görlich D. Rapoport T.A. Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 14Oliver J. Jungnickel B. Görlich D. Rapoport T. High S. FEBS Lett. 1995; 362: 126-130Crossref PubMed Scopus (55) Google Scholar). In the minimal system, the translocation machinery is comprised of the signal recognition particle receptor which functions in the targeting of ribosome/nascent chain complexes to the ER, the Sec61p complex, which is thought to serve as a ribosome receptor and translocation channel, and, in some instances, the integral membrane protein TRAM, which participates in signal sequence recognition (13Görlich D. Rapoport T.A. Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 14Oliver J. Jungnickel B. Görlich D. Rapoport T. High S. FEBS Lett. 1995; 362: 126-130Crossref PubMed Scopus (55) Google Scholar, 15Kalies K.U. Gorlich D. Rapoport T.A. J. Cell Biol. 1994; 126: 925-934Crossref PubMed Scopus (146) Google Scholar). The identification of the Sec61p complex as the primary ribosome receptor and translocation channel suggests that ribosome association with Sec61p could provide the aqueous pathway for nascent chain transit into the lumen (15Kalies K.U. Gorlich D. Rapoport T.A. J. Cell Biol. 1994; 126: 925-934Crossref PubMed Scopus (146) Google Scholar, 16Crowley K.S. Reinhart G.D. Johnson A.E. Cell. 1993; 73: 1101-1115Abstract Full Text PDF PubMed Scopus (237) Google Scholar). is molecular and studies that proteins an in protein translocation across the ER and the membrane J.P. Rose M.D. J. Cell Biol. 1990; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Schekman J. Cell Biol. 1993; PubMed Scopus Google Scholar, S. S. Rapoport T.A. 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The of the of protein translocation is by the that protein and folding are coincident and that protein folding in an the ER which is in molecular chaperones and protein folding To the contribution of protein folding and lumenal chaperone-nascent chain interactions to the translocation of wild type and mutant forms of preprolactin were in in vitro. The mutant preprolactin used in contains an 18-amino acid substitution at the terminus of the mature protein, eliminating a disulfide-bonded loop C.S. S.M. Rev. 7: PubMed Scopus Google Scholar, T.A. PubMed Scopus Google Scholar). wild type prolactin was efficiently the folding mutant accumulated in the ER as protein-protein and disulfide-bonded aggregates. In vitro, both and forms of prolactin were translocated with were recovered as aggregates. aggregation was co-translationally. In the absence of lumenal formation was and was by a in translocation the of these that lumenal chain interactions are to translocation and in protein-protein aggregation may serve as the predominant protein folding end point. COS-7 Cell. Full Text PDF PubMed Scopus Google were in with and were performed by the as in J. Biol. 1993; Full Text PDF PubMed Google was by of a preprolactin the T. J. Cell Biol. PubMed Scopus Google with and and of the mammalian was by as In the mutant FA, the amino of mature prolactin are: whereas in wild type the sequence In wild type a disulfide bond is between amino and T.A. PubMed Scopus Google Scholar). In the mutant the disulfide-bonded loop with a In the of products were the and and and were gel and used as in a with and was performed as and the gel was with and into the for identified by and were to to to the of the studies were performed as in A.J. 1990; PubMed Scopus Google in were in and and the amino acid by in and for at The was performed by addition of in of for at the was and in the indicated the was and the by into were in and by addition of of a were with to a of and to were by addition of of a of the of and performed by addition of prolactin S. and at complexes were by addition of of a of and at for was by with and in prolactin was by addition of of and for at and at were on as C.V. J. Cell Biol. 1995; PubMed Scopus Google Scholar). In experiments in which disulfide bond formation was were in PBS, and to free by addition of for on as in Helenius J. Helenius A. Nature. 1992; PubMed Scopus Google COS-7 were as were as the and the recovered by into The were in to on for and with a tight The was to and for at The was and the The was for at in the The the was in to a of were performed on for with was at were by addition of to and a on the were with and prolactin translation products by studies were performed as and by addition of with Cell were as Helenius J. Helenius A. Nature. 1992; PubMed Scopus Google and of by for at were in with were and with a were for at in the were and prolactin performed as In experiments were performed as in C.V. Blobel G. J. Cell Biol. 1989; PubMed Scopus Google were performed as in T. PubMed Scopus Google Scholar, and rough as in P. Blobel G. PubMed Scopus Google of translocation rough was performed as in C. G. Blobel G. Cell Biol. PubMed Scopus Google The are of a of that the and C.S. S.M. Rev. 7: PubMed Scopus Google Scholar, T.A. PubMed Scopus Google Scholar). 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Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). are in and of In native and RM, both were to efficiently translocation to the of signal sequence and a of that the the as efficiently as and is in the studies in B. In the in membrane were the translation by and the and for the of associated translation products. In the absence of and the nascent is recovered in the whereas in the of native the signal mature with membrane In are performed in the of of the mature protein are recovered in the and The of the of prolactin in the that the targeting and translocation to the of signal sequence of the free the to the and was thus recovered in the The structural of the prolactin in the of and was be and with in the of RM, is recovered the with the predominant at the of the in the of RM, was recovered in the with a to that for the folding mutant in vivo andin vitro In in the of the translocation and structural of the and were that in the absence of lumenal the protein folding and translocation are the in vivo in the protein that protein translocation in the mammalian the process is Current models suggest of In model, the association of the ribosome with the protein conducting channel is thought to provide a pathway for the nascent chain such that the nascent chain alternative into the ER in the of membrane into the ER (1Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (708) Google T.A. Jungnickel B. Katay U. Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (491) Google Scholar). In model, the of the nascent chain the protein conducting channel serve as the although protein translocation in the mammalian ER may be driven in a to that for translocation in ER as well as for protein into (3Neupert W. Hartl F.-U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 5Nicchitta C.V. Blobel G. Cell. 1993; 73: 989-998Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 6Sanders S.L. Whitfield K.M. Vogel J.P. Rose M.D. Schekman R.W. Cell. 1992; 69: 353-365Abstract Full Text PDF PubMed Scopus (273) Google Scholar, C.V. Semin. Cell. Dev. Biol. 1996; 7: 497-503Crossref Scopus (3) Google Scholar, J. Schekman Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In the latter model, the free energy for transport is through interactions of the nascent chain with molecular chaperones and structural in the nascent chain that translocation in the that such interactions a and bias the of the nascent chain to vectorial transport (3Neupert W. Hartl F.-U. Craig E.A. Pfanner N. Cell. 1990; 63: 447-450Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 4Simon S.M. Peskin C.S. Oster G.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3770-3774Crossref PubMed Scopus (361) Google Scholar, 5Nicchitta C.V. Blobel G. Cell. 1993; 73: 989-998Abstract Full Text PDF PubMed Scopus (153) Google Scholar). folding in the ER coincident with is by a free energy can be through in the primary protein and in an the ER which is in molecular chaperones (7Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (773) Google Scholar, 9Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (557) Google Scholar, 10Rothman J.E. Cell. 1989; 59: 591-601Abstract Full Text PDF PubMed Scopus (626) Google Scholar, 11Gething M.-J. Sambrook J. Nature. 1992; 355: 33-47Crossref PubMed Scopus (3565) Google Scholar, Rev. Cell Biol. 1989; 5: PubMed Scopus Google Scholar). is with these in that the of a protein folding pathway on ER translocation were in vivo and in vitro. folding was through of the folding as well as by of the ER lumenal The folding used was the protein which contains a disulfide-bonded loop at the terminus of the mature protein T.A. PubMed Scopus Google Scholar). of prolactin is the to be translocated into the ER lumen and the terminus of the folding domain. was that the folding pathway and interaction with lumenal chaperones and protein folding be significantly at an of the translocation In in vivo was that the mutant prolactin an comprised of protein-protein and disulfide and was type in was efficiently with a of was efficiently of the cells, and of the was to the these that although the protein folding pathway significantly protein translocation is with a of observations and that across the ER membrane the nascent chain in an and an the ER lumen J. Biol. Full Text PDF PubMed Google Scholar, M.-J. Sambrook J. Cell. Full Text PDF PubMed Scopus Google Scholar, Rose J. Biol. Full Text PDF PubMed Google Scholar, C. C. J. Cell Biol. Scopus Google Scholar). In the of translocation was that protein as a could a to protein at to chain in the proteins and T. Helenius A. Cell Biol. 1992; PubMed Scopus (153) Google Scholar). protein-protein aggregation to to the of occur co-translationally. of preprolactin proteins in the of rough was that aggregation can In these forms of to translocation intermediates T. J. Cell Biol. PubMed Scopus Google Scholar, C.V. Blobel G. J. Cell Biol. 1989; PubMed Scopus Google Scholar, B. Rapoport T.A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, T. P. J. Cell Biol. 1989; PubMed Scopus Google Scholar), were into rough and the structural of the nascent by of a protein-protein interactions are to occur (7Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (773) Google may be to J.E. J. Cell Sci. 1995; Google is that the for the prolactin intermediates interactions of the nascent with lumenal native proteins which in the lumen and membrane have as to interactions with the ER lumenal proteins suggests that the latter is observations were in the in vitro In studies with the and translation was that translocation a of the WT, and the of the of were recovered as heterogeneous aggregates. although the folding could be vitro, was also that in vitro the protein in lumenal the efficiency of translocation and folding were the of the lumenal chaperones on the efficiency of the of i.e. translocation to and signal sequence C.V. Semin. Cell. Dev. Biol. 1996; 7: 497-503Crossref Scopus (3) Google Scholar, C.V. Blobel G. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). in the of rough lumenal the of the translation products were as aggregates. The that prolactin could be recovered in the of the that a of the prolactin retrograde transport the and also be that the structural of in the lumenal was to that of the folding FA, in the of native membranes. These suggest that the lumenal proteins through interactions with the nascent chain in lumenal proteins may transport and interactions of the lumenal chaperones with the nascent aggregation and enhance the efficiency of protein The have ramifications on investigations into the molecular mechanism of protein To the molecular of is that the the in vivo In many the in of the structural of the nascent that in vitro, translocated proteins may folding and as protein-protein and disulfide-bonded aggregates. such in protein folding are such as in which the lumenal of chaperones and protein folding are is for studies of the of translocation for which for lumenal proteins in translocation have identified the of these suggest that the for protein translocation be to protein folding and for and of the
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