A 56-kDa Selenium-binding Protein Participates in Intra-Golgi Protein Transport

Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors. By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity. Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56. Recombinant SBP56 expressed inEscherichia coli exhibited transport activity when added to the cell-free intra-Golgi transport. Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transportin vitro. Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes. Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane. The experiments presented in this study indicate that SBP56 participates in late stages of intra-Golgi protein transport. Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors. By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity. Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56. Recombinant SBP56 expressed inEscherichia coli exhibited transport activity when added to the cell-free intra-Golgi transport. Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transportin vitro. Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes. Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane. The experiments presented in this study indicate that SBP56 participates in late stages of intra-Golgi protein transport. Coat protein N-ethylmaleimide NEM-sensitive fusion protein soluble NSF attachment protein SNAP receptors dithiothreitol vesicular stomatitis virus Chinese hamster ovary brefeldin A guanosine 5′-O-thiotriphosphate selenium-binding protein polyacrylamide gel electrophoresis nickel-nitrilotriacetic acid protein disulfide isomerase phosphoglucomutase Vesicles that bud from a donor membrane compartment and then dock and fuse with an acceptor membrane mediate transport of proteins between different organelles of eukaryotic cells (1.Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2354) Google Scholar, 2.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar). 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The intra-Golgi transport of of a of donor and acceptor Golgi of of cytosolic of cytosolic of of of p115, and and for the transport activity of different cytosolic added to the The transport at for protein was as previously A. A. Elazar Z. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the transport experiments shown in this study at in in the of cytosolic was from For the assay, as by M. P. P. Biol. Cell. 1994; PubMed Scopus Google Scholar). Golgi from cells with the donor with for on at was added to The was under for the transport or the with of bovine brain cytosol, the cytosol was to by with was added a of at was The was for and in a at for The was in of and and the was by in a at for This was The was to with and at a with The was with of and the was This was The of this was for the cytosolic The was in a at for and then at a in The was with of the and the was This a protein of a protein in transport was purified from the of this A. A. Elazar Z. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Elazar Z. EMBO J. PubMed Scopus Google Scholar). The was for the cytosolic The was and in a at for at The and This protein of The of was to that of by with the to a of was then at a and in The was with of the and proteins with a of in of and for transport activity using the transport significant intra-Golgi transport at and This protein of The of was to by the of and the was to by was then at in The was with of and proteins with a of to in by of of and for transport activity using the transport transport at and This protein of was and at in the The was with of and then to a from to by a of of and for transport activity using the transport transport at and This protein of the acid of the proteins in on protein in and the by with at the the the For electrophoresis was on polyacrylamide proteins in bovine and in The of antibodies and for are specifically for each antibodies by or by on polyacrylamide by with The SBP56 in was as a from the of P. R. the for The SBP56 was using in the coli by the was in of and at to an A of with for and in at for The was in a and A. cells by the and by at for using was a nickel-nitrilotriacetic acid and with of and protein was by of of SBP56 and and in at SBP56 was purified as and to polyclonal antibodies in by the at the of Science. anti-SBP56 polyclonal antibodies on a to antibodies and on The was then to using at anti-SBP56 polyclonal antibodies to protein and to the by cytosol was with and of SBP56 was by two with protein per of cytosol proteins per each for by at as previously R.H. Biol. 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By using anti-SBP56 antibodies we that SBP56 is predominantly in the cytosolic a small amount of SBP56 was in the cytosolic may for the significant in the SBP56 is in intra-Golgi transport, we and expressed a of this protein and its activity in the transport For that of SBP56 was a to a protein with at its The protein was expressed in E. coli and purified on a The SBP56 was for its to in the transport of the transport the to for this activity The activity of purified or in with the that transport factors. the of the SBP56 in the intra-Golgi transport we reconstituted the transport with this of of anti-SBP56 antibodies inhibited to of the transport activity was with the the transport activity was to its for the transport assay, by the anti-SBP56 of transport was only when the anti-SBP56 antibodies added the of transport. the of SBP56 in intra-Golgi transport, the cytosol was of SBP56 by anti-SBP56 anti-SBP56 to of the SBP56 from the cytosol The of SBP56 from the cytosol was by in intra-Golgi transport of to the cytosol the transport activity experiments for the of SBP56 in intra-Golgi transport. two to the that the of the by SBP56 from activity of of transport. a by and co-workers M. P. P. Biol. Cell. 1994; PubMed Scopus Google Scholar). Golgi from cells with the protein and are in the this system the of to protein is of membrane fusion M. P. P. Biol. Cell. 1994; PubMed Scopus Google Scholar). the cytosol is required to the to the to the transport shown in the transport assay, whereas of to the the cytosolic SBP56 anti-SBP56 to of the transport assay, the in the the that SBP56 is in the step of the transport 1992; PubMed Scopus Google Scholar), we a in we the of and anti-SBP56 antibodies the in the transport was at different by or each of the anti-SBP56 or was added at the the was to for a of the at a may indicate a of events of the intra-Golgi transport only at the of the and as when added at the of the of transport. The to the anti-SBP56 antibodies when added transport was This that SBP56 is prior to the step by the the transport to the anti-SBP56 antibodies the the that SBP56 to vesicle GTPases of the shown to in intra-Golgi transport in vitro T. N. Elazar Z. J. Biol. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. T. Rothman J.E. J. Biol. 1994; Full Text PDF PubMed Google Scholar, H. S. R. J. Cell Biol. 1991; PubMed Scopus Google Scholar). added in in of proteins from intracellular H. K. K. T. Zerial M. J. Biol. 1993; Full Text PDF PubMed Google Scholar) of intra-Golgi transport Z. T. Rothman J.E. J. Biol. 1994; Full Text PDF PubMed Google Scholar). has been recently that specifically vesicle docking fusion with the target membrane X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (292) Google Scholar). the we to the at SBP56 is the of intra-Golgi transport by or anti-SBP56 antibodies was this we a transport in Golgi with different for under transport and then and for transport activity in the of shown in of cytosol to Golgi that previously with anti-SBP56 or to of the intra-Golgi transport. the of a transport in Golgi with the for and then and with cytosol in the or of the antibodies to the transport activity of Golgi that with that proteins act SBP56. a we that the transport activity of Golgi that with anti-SBP56 antibodies experiments that SBP56 to proteins in cell-free intra-Golgi transport, its in late stages of the transport. Following SBP56 was in the soluble with only a small associated with the the of the was on two at the to and the of a polyclonal M. Orci L. Ravazzola M. Amherdt M. L. P. Rothman J.E. Söllner T.H. J. Cell Biol. 1996; PubMed Scopus Google Scholar, F. R. W. Science. 1996; 272: PubMed Scopus Google or antibodies to of the different obtained from the SBP56 with to the Golgi to a with in of the ER was as and in the cytosolic in the to the ER and the Golgi that SBP56 was specifically associated with membranes. The that only a small of SBP56 was to that this protein is only associated with the Golgi membranes. that SBP56 well with the at that are for intra-Golgi transport in to SBP56 from the with of to whereas with SBP56 from the with SBP56 to the soluble experiments that of SBP56 is peripherally associated with the membrane. By using Golgi and we that this protein specifically to the is the well characterized cell-free that reconstitutes intra-Golgi transport to and a 56-kDa protein from bovine brain cytosol and its significant transport activity. The protein is to a selenium-binding protein of was previously as a protein that M.P. 1989; 10: PubMed Scopus Google Scholar, M.P. T. J. R. PubMed Scopus Google Scholar), its has been several of that SBP56 in vesicular the protein was on the basis of a transport expressed in E. coli is in the cell-free transport antibodies specifically the transport cytosol of SBP56 is in intra-Golgi and the membrane of SBP56 is associated with the we that SBP56 is in the transport process in vitro in the by the activity of or the cell-free transport assay, to a the cytosolic This the of transport in the for from fractionation in we transport that of SBP56. and Z. to SBP56 is a highly protein with in and the acid of SBP56 shows significant to yeast This is transport are highly between yeast and The yeast may a homologue of SBP56 that fulfills its SBP56 may as a of a step in transport that is in or its is SBP56 was as a cytosolic protein that M.P. T. J. R. PubMed Scopus Google Scholar). This protein is highly expressed in and and has been suggested that its with of in M. 1998; 58: Google Scholar). the of this protein has been the by to SBP56 the of is we that SBP56 in vesicular transport. between and the activity of this protein in the transport may well that this protein has has been suggested that proteins are required to the SNARE complex formation S.K. Lupashin V.V. Schmitt H.D. Waters M.G. J. Cell Biol. 1996; 132: 755-767Crossref PubMed Scopus (159) Google Scholar, M. Tani K. S. P. T. Rothman J.E. Söllner T. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), NSF and SNAP (30.Ungermann C. Sato K. Wickner W. Nature. 1998; 396: 543-548Crossref PubMed Scopus (279) Google A. Wickner W. A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). using the yeast in vitro system has been recently that proteins are required for docking of vesicles to the Golgi membrane and for fusion X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (292) Google Scholar). Based on the in we that SBP56 is of the proteins. that SBP56 is in docking or the by Rothman and (27.Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar), that the minimal that fusion between vesicle and its we that SBP56 with soluble to and this several cytosolic based on activity in the intra-Golgi cell-free transport NSF and are to act in and the fusion (25.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, A. Wickner W. A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar); p115, a that between COPI vesicles and the Golgi (18.Sonnichsen B. Lowe M. Levine T. Jamsa E. Dirac-Svejstrup B. Warren G. J. Cell Biol. 1998; 140: 1013-1021Crossref PubMed Scopus (254) Google Scholar); A. A. Elazar Z. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), together with NSF and SNAP in the Golgi A. A. Elazar Z. EMBO J. PubMed Scopus Google Scholar); Waters M.G. 1998; PubMed Scopus Google Scholar); and an 13 complex that at late stages of the intra-Golgi transport Waters M.G. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins that between the membrane and the cytosol F. S.R. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) are required in the cytosolic of the cell-free transport This is to a of in the membrane we SBP56 as soluble protein in transport. that this protein may required at late stages of transport, docking and we the at SBP56 is required or the activity of of the transport factors. The in the to intra-Golgi transport in vitro with a well defined set of cytosolic and peripheral membrane proteins. J. E. Rothman for and W. for the and P. R. for the of SBP56. for are to the of for