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Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

2010; Elsevier BV; Volume: 285; Issue: 20 Linguagem: Inglês

10.1074/jbc.m109.086272

1083-351X

Yoichi Noda, Koji Yoda,

Photosynthetic Processes and Mechanisms

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Δsvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26. Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Δsvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26. IntroductionThe Golgi apparatus is comprised of multiple compartments with different components and functions. These compartments are successively formed by the maturation of the earliest compartment derived from the endoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumHAhemagglutininGSTglutathione S-transferasemAbmonoclonal antibodyGMPPNPguanosine 5′-(β,γ-imido)triphosphateGDPβSguanosine 5′-(β-thio)diphosphate. through homotypic fusion of the COPII vesicles (1Pelham H.R. Trends Cell Biol. 1998; 8: 45-49Abstract Full Text PDF PubMed Scopus (99) Google Scholar). The maturation of compartments is believed to occur by removing the components of the earlier compartments and acquiring those of the later compartments in a COPI vesicle-dependent manner (2Losev E. Reinke C.A. Jellen J. Strongin D.E. Bevis B.J. Glick B.S. Nature. 2006; 441: 1002-1006Crossref PubMed Scopus (276) Google Scholar, 3Matsuura-Tokita K. Takeuchi M. Ichihara A. Mikuriya K. Nakano A. Nature. 2006; 441: 1007-1010Crossref PubMed Scopus (264) Google Scholar). The newly synthesized Golgi proteins are first translocated in the ER and then selectively incorporated in the COPII vesicles and transported to the earliest Golgi compartment or the cis-Golgi network. When the small G-protein Sar1 is activated by Sec12 on the ER, Sec23/Sec24 subunits are recruited to form the prebudding complex containing the selected cargo proteins (4Kuehn M.J. Herrmann J.M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (322) Google Scholar). Successive binding of Sec13/Sec31 subunits induces ER membrane deformation, budding, and release of the COPII vesicles (5Salama N.R. Yeung T. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). The retrieval of the Golgi proteins from the compartment at a certain maturation stage is carried out by the COPI vesicles after activation of the G-protein Arf1 and recruitment of coatomer proteins, and the selected cargos are transported to the earlier compartments or to the ER (6Gaynor E.C. Graham T.R. Emr S.D. Biochim. Biophys. Acta. 1998; 1404: 33-51Crossref PubMed Scopus (76) Google Scholar, 7Todorow Z. Spang A. Carmack E. Yates J. Schekman R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13643-13648Crossref PubMed Scopus (43) Google Scholar).The first carbohydrates of glycoproteins are transferred from the dolichol intermediates to certain Asn or Ser/Thr residues of the precursor polypeptides in the ER, and then further glycosyl residues are added or modified after moving from the ER to the Golgi (8Dean N. Biochim. Biophys. Acta. 1999; 1426: 309-322Crossref PubMed Scopus (157) Google Scholar). The enzymatic specificity of the glycosyltransferases and their localization in the Golgi compartments correlate with the successive addition of sugar residues. Each Golgi enzyme is attributed to a certain specific compartment through the dynamic maturation process by the exit and entry of selected components in the COPI vesicles. Therefore, the selective incorporation of the cargo proteins in the COPII and COPI vesicles should be very important for maintaining the specific protein composition of Golgi compartments. The motif sequences such as LXX(L/M)E for COPII selection (9Mossessova E. Bickford L.C. Goldberg J. Cell. 2003; 114: 483-495Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 10Miller E.A. Beilharz T.H. Malkus P.N. Lee M.C. Hamamoto S. Orci L. Schekman R. Cell. 2003; 114: 497-509Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) or K(X)KXX for COPI selection (11Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar, 12Letourneur F. Gaynor E.C. Hennecke S. Démollière C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (664) Google Scholar) are found to interact directly with the coat subunits when the motif-containing proteins are incorporated into the vesicles. In addition to the direct selective interaction with the coat subunits (13Springer S. Schekman R. Science. 1998; 281: 698-700Crossref PubMed Scopus (164) Google Scholar), incorporation in the vesicles via interaction with adaptor components has been reported (14Sato K. Nakano A. FEBS Lett. 2007; 581: 2076-2082Crossref PubMed Scopus (154) Google Scholar). A peripheral membrane protein, Vps74, was found to bind to the N-terminal cytoplasmic domain of a subset of mannosyltransferases and is required to maintain their Golgi localization (15Schmitz K.R. Liu J. Li S. Setty T.G. Wood C.S. Burd C.G. Ferguson K.M. Dev. Cell. 2008; 14: 523-534Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 16Tu L. Tai W.C. Chen L. Banfield D.K. Science. 2008; 321: 404-407Crossref PubMed Scopus (173) Google Scholar). However, we have only limited knowledge about the selective transport mechanism of the Golgi glycosyltransferases.Svp26 was discovered as a novel function-unknown protein in our global inspection of membrane proteins in the early Golgi compartment (17Inadome H. Noda Y. Adachi H. Yoda K. Mol. Cell Biol. 2005; 25: 7696-7710Crossref PubMed Scopus (43) Google Scholar). Svp26 is an integral membrane protein with four predicted transmembrane segments. Although Svp26 is dispensable for viability of the yeast, we found abnormal hypermannosylation of N-glycosyl chains and mislocalization of Golgi mannosyltransferase Ktr3 to the ER in the Δsvp26 disruptant cell. Immunoprecipitation from the cleared lysate using 1% Triton X-100 indicated specific binding between Ktr3 and Svp26. These results suggest that Svp26 functions to ensure the correct localization of Ktr3 in the Golgi compartments.We examined whether other mannosyltransferases depend on Svp26 to localize in the Golgi compartments. In this paper, we report that several mannosyltransferases behave as Ktr3 does, and Svp26 helps the selective incorporation of these mannosyltransferases into the COPII vesicles. The lumenal domains of the cargo are responsible for Svp26 dependence.DISCUSSIONThe Golgi O-mannosyltransferase Ktr3 is found in the ER in the Δsvp26 cells. Here, we examined whether the Golgi localization of the five N-mannosyltransferases, Och1, Mnn1, Mnn2, Mnn5, and Mnn9 are also Svp26-dependent. Mnn2 and Mnn5, like Ktr3, are found in the ER in the absence of Svp26. The Golgi localization of Mnn1 and Mnn9 is not affected. A small amount of Och1 was observed in the ER in half of the cells. We tested whether any of the five N-mannosyltransferases bind to Svp26 by immunoprecipitation from the cleared lysate containing 1% digitonin. Although co-immunoprecipitation from such lysate could not exclude indirect interaction because digitonin leaves many membrane protein complexes intact, Svp26 was not detected in the immunoprecipitate of Mnn1, the Golgi localization of which is independent of Svp26. On the other hand, a significant amount of Svp26 was co-immunoprecipitated with Mnn2 and Mnn5, which suggests that their Golgi localization and ability to interact with Svp26 are somehow related. These interactions are likely to be weaker than the Ktr3-Svp26 interaction, as no Svp26 interaction was detected when using Triton X-100 solubilized membranes. A small amount of Svp26 was detected in the immunoprecipitate of Och1 that shows weak ER mislocalization in the Δsvp26 disruptant. A small amount of Svp26 was also found in the immunoprecipitate of Mnn9, although the Golgi localization of Mnn9 is not affected by the loss of Svp26. Mnn9 exists in the stable complex with Van1 (M-pol I, V-complex) or with four proteins including Anp1 (M-pol II, A-complex) (18Hashimoto H. Yoda K. Biochem. Biophys. Res. Commun. 1997; 241: 682-686Crossref PubMed Scopus (35) Google Scholar, 23Jungmann J. Munro S. EMBO J. 1998; 17: 423-434Crossref PubMed Scopus (184) Google Scholar). This complex formation may be more important for the Golgi localization of Mnn9 than its binding to Svp26, or there may be other proteins that bind to and assist the exit of other subunits of the complex, which function redundantly with Svp26 in facilitating the entry of the Mnn9-containing complex into COPII vesicles.Our in vitro budding experiment suggested that the selective incorporation of Ktr3 and Mnn2 in the COPII vesicles is clearly stimulated by Svp26. At the very least, the incorporation of cargo into the COPII vesicle is a stage of Svp26 dependence for Ktr3 and Mnn2. The ER membrane fraction of the Δsvp26 disruptant contains a larger amount of these mannosyltransferases than the wild-type ER fraction (Fig. 5). It is likely that the efficiency of the exit from the ER is retarded in the Δsvp26 disruptant, and Ktr3 and Mnn2 accumulate in the ER. Svp26 is a good cargo for COPII vesicles and efficiently exits from the ER in the in vitro budding assay (data not shown) (25Bue C.A. Bentivoglio C.M. Barlowe C. Mol. Biol. Cell. 2006; 17: 4780-4789Crossref PubMed Scopus (29) Google Scholar). Ktr3, Mnn2, and Mnn5 can bind to Svp26. It is most probable that the interaction with Svp26 physically stimulates their incorporation into the COPII vesicles and exit from the ER.It is reported that certain sequence motifs of the cargo molecules are recognized by the Sec24 subunit of the COPII coat (29Votsmeier C. Gallwitz D. EMBO J. 2001; 20: 6742-6750Crossref PubMed Scopus (107) Google Scholar). These motifs are, for example, found in the cytoplasmic domain of soluble NSF attachment protein receptor (SNARE) proteins (9Mossessova E. Bickford L.C. Goldberg J. Cell. 2003; 114: 483-495Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 10Miller E.A. Beilharz T.H. Malkus P.N. Lee M.C. Hamamoto S. Orci L. Schekman R. Cell. 2003; 114: 497-509Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Also, because Svp26 is efficiently incorporated in the prebudding complex, it may have some sequence motif or structural characteristics that will be recognized by the coat subunits. The Svp26-dependent Golgi mannosyltransferases may therefore indirectly contact with the coat proteins through the direct interaction with Svp26. In other words, Svp26 mediates the interaction between the mannosyltransferase cargo and coat proteins. Therefore, we propose Svp26 acts as a sorting adaptor for COPII coats. Recently, it has been reported that the exit of the vacuolar alkaline phosphatase precursor proPho8 from the ER via COPII vesicle depends on Svp26/Erv26 (30Bue C.A. Barlowe C. J. Biol. Chem. 2009; 284: 24049-24060Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 31Anand V.C. Daboussi L. Lorenz T.C. Payne G.S. Mol. Biol. Cell. 2009; 20: 1592-1604Crossref PubMed Scopus (34) Google Scholar, 32Dancourt J. Barlowe C. Traffic. 2009; 10: 1006-1018Crossref PubMed Scopus (12) Google Scholar). Investigation of this process revealed that Svp26 acts as the adaptor or receptor for the COPII coat. Also, another type II membrane protein, Gda1, which is a guanosine diphosphatase located in the Golgi, is thought to be dependent on Svp26 for its exit from the ER (31Anand V.C. Daboussi L. Lorenz T.C. Payne G.S. Mol. Biol. Cell. 2009; 20: 1592-1604Crossref PubMed Scopus (34) Google Scholar). It is possible that some other proteins with little or no affinity to the coat subunits utilize Svp26 as the adaptor for the COPII coat to efficiently exit the ER. The presence of adaptors for coat proteins is important for accepting a wide variety of proteins into the COPII coat.Svp26 binds to proteins with different molecular masses and with little sequence homology and stimulates the exit of the interacting proteins from the ER. The nature of the interaction between Svp26 and its interacting proteins is largely unknown. However, by the domain exchanging of Svp26-dependent Ktr3 or Mnn2 and Svp26-independent Mnn1, we found the lumenal domains are responsible for the dependence on Svp26 for ER exit. Recently, it has been reported that the lumenal domain of proPho8 interacts with Svp26 (32Dancourt J. Barlowe C. Traffic. 2009; 10: 1006-1018Crossref PubMed Scopus (12) Google Scholar). It may be a common feature that Svp26 recognizes the lumenal domains of its interacting proteins.As the lumenal domain of mannosyltransferase is believed to function as the catalytic domain of the enzyme (28Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7945-7950Crossref PubMed Scopus (315) Google Scholar), it is very important when and how the interaction with Svp26 ends after the mannosyltransferase arrives in the Golgi compartment and what effect the interaction with Svp26 may have on the enzyme activity. The N-glycosyl chains of invertase and the modified hen egg lysozyme accept hypermannosylation in the Δsvp26 disruptant (17Inadome H. Noda Y. Adachi H. Yoda K. Mol. Cell Biol. 2005; 25: 7696-7710Crossref PubMed Scopus (43) Google Scholar), the mechanism of which is completely unknown. If Svp26 acts not only as the sorting adaptor for COPII coats but also as a negative regulator of the enzyme activity to control the degree of mannosylation in the early Golgi, the mannosyltransferases that arrive in the Golgi by a default transport pathway may cause the addition of more mannose in the Δsvp26 disruptant.Even in the absence of Svp26, a small amount of Svp26-dependent mannosyltransferase is delivered in the Golgi by the "default" traffic pathway. This may mean that COPII vesicles nonselectively incorporate components in the ER. Alternatively, there may be a COPII-independent delivery of proteins from the ER to Golgi. A small amount of Mnn2 is released from the ER-enriched fraction even without the addition of purified COPII subunits for an unknown reason (Fig. 4B). This phenomenon is more significant in the Mnn2OP membrane. Interestingly, this release without the addition of the COPII component was significantly decreased in the Δsvp26 ER fraction that has similarly accumulated Mnn2. Therefore, the uncharacterized delivery mechanism, if it exists, seems to also be Svp26-dependent in the case of Mnn2. IntroductionThe Golgi apparatus is comprised of multiple compartments with different components and functions. These compartments are successively formed by the maturation of the earliest compartment derived from the endoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumHAhemagglutininGSTglutathione S-transferasemAbmonoclonal antibodyGMPPNPguanosine 5′-(β,γ-imido)triphosphateGDPβSguanosine 5′-(β-thio)diphosphate. through homotypic fusion of the COPII vesicles (1Pelham H.R. Trends Cell Biol. 1998; 8: 45-49Abstract Full Text PDF PubMed Scopus (99) Google Scholar). The maturation of compartments is believed to occur by removing the components of the earlier compartments and acquiring those of the later compartments in a COPI vesicle-dependent manner (2Losev E. Reinke C.A. Jellen J. Strongin D.E. Bevis B.J. Glick B.S. Nature. 2006; 441: 1002-1006Crossref PubMed Scopus (276) Google Scholar, 3Matsuura-Tokita K. Takeuchi M. Ichihara A. Mikuriya K. Nakano A. Nature. 2006; 441: 1007-1010Crossref PubMed Scopus (264) Google Scholar). The newly synthesized Golgi proteins are first translocated in the ER and then selectively incorporated in the COPII vesicles and transported to the earliest Golgi compartment or the cis-Golgi network. When the small G-protein Sar1 is activated by Sec12 on the ER, Sec23/Sec24 subunits are recruited to form the prebudding complex containing the selected cargo proteins (4Kuehn M.J. Herrmann J.M. Schekman R. Nature. 1998; 391: 187-190Crossref PubMed Scopus (322) Google Scholar). Successive binding of Sec13/Sec31 subunits induces ER membrane deformation, budding, and release of the COPII vesicles (5Salama N.R. Yeung T. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). The retrieval of the Golgi proteins from the compartment at a certain maturation stage is carried out by the COPI vesicles after activation of the G-protein Arf1 and recruitment of coatomer proteins, and the selected cargos are transported to the earlier compartments or to the ER (6Gaynor E.C. Graham T.R. Emr S.D. Biochim. Biophys. Acta. 1998; 1404: 33-51Crossref PubMed Scopus (76) Google Scholar, 7Todorow Z. Spang A. Carmack E. Yates J. Schekman R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13643-13648Crossref PubMed Scopus (43) Google Scholar).The first carbohydrates of glycoproteins are transferred from the dolichol intermediates to certain Asn or Ser/Thr residues of the precursor polypeptides in the ER, and then further glycosyl residues are added or modified after moving from the ER to the Golgi (8Dean N. Biochim. Biophys. Acta. 1999; 1426: 309-322Crossref PubMed Scopus (157) Google Scholar). The enzymatic specificity of the glycosyltransferases and their localization in the Golgi compartments correlate with the successive addition of sugar residues. Each Golgi enzyme is attributed to a certain specific compartment through the dynamic maturation process by the exit and entry of selected components in the COPI vesicles. Therefore, the selective incorporation of the cargo proteins in the COPII and COPI vesicles should be very important for maintaining the specific protein composition of Golgi compartments. The motif sequences such as LXX(L/M)E for COPII selection (9Mossessova E. Bickford L.C. Goldberg J. Cell. 2003; 114: 483-495Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 10Miller E.A. Beilharz T.H. Malkus P.N. Lee M.C. Hamamoto S. Orci L. Schekman R. Cell. 2003; 114: 497-509Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) or K(X)KXX for COPI selection (11Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (480) Google Scholar, 12Letourneur F. Gaynor E.C. Hennecke S. Démollière C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (664) Google Scholar) are found to interact directly with the coat subunits when the motif-containing proteins are incorporated into the vesicles. In addition to the direct selective interaction with the coat subunits (13Springer S. Schekman R. Science. 1998; 281: 698-700Crossref PubMed Scopus (164) Google Scholar), incorporation in the vesicles via interaction with adaptor components has been reported (14Sato K. Nakano A. FEBS Lett. 2007; 581: 2076-2082Crossref PubMed Scopus (154) Google Scholar). A peripheral membrane protein, Vps74, was found to bind to the N-terminal cytoplasmic domain of a subset of mannosyltransferases and is required to maintain their Golgi localization (15Schmitz K.R. Liu J. Li S. Setty T.G. Wood C.S. Burd C.G. Ferguson K.M. Dev. Cell. 2008; 14: 523-534Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 16Tu L. Tai W.C. Chen L. Banfield D.K. Science. 2008; 321: 404-407Crossref PubMed Scopus (173) Google Scholar). However, we have only limited knowledge about the selective transport mechanism of the Golgi glycosyltransferases.Svp26 was discovered as a novel function-unknown protein in our global inspection of membrane proteins in the early Golgi compartment (17Inadome H. Noda Y. Adachi H. Yoda K. Mol. Cell Biol. 2005; 25: 7696-7710Crossref PubMed Scopus (43) Google Scholar). Svp26 is an integral membrane protein with four predicted transmembrane segments. Although Svp26 is dispensable for viability of the yeast, we found abnormal hypermannosylation of N-glycosyl chains and mislocalization of Golgi mannosyltransferase Ktr3 to the ER in the Δsvp26 disruptant cell. Immunoprecipitation from the cleared lysate using 1% Triton X-100 indicated specific binding between Ktr3 and Svp26. These results suggest that Svp26 functions to ensure the correct localization of Ktr3 in the Golgi compartments.We examined whether other mannosyltransferases depend on Svp26 to localize in the Golgi compartments. In this paper, we report that several mannosyltransferases behave as Ktr3 does, and Svp26 helps the selective incorporation of these mannosyltransferases into the COPII vesicles. The lumenal domains of the cargo are responsible for Svp26 dependence.