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Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex.

Otte S, Belden WJ, Heidtman M, Liu J, Jensen ON, Barlowe C - J. Cell Biol. (2001)

Bottom Line: The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain.When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains.A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

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Erv41p and Erv46p depend on each other for their expression. wt, wild-type strain FY834; 46Δ, erv46Δ deletion strain CBY799; 41Δ, erv41Δ deletion strain CBY797; and 41Δ 46Δ, erv41Δ erv46Δ double deletion strain CBY795. Semiintact cells were resolved on a 12.5% polyacrylamide gel and immunoblotted with polyclonal anti-Erv41p and anti-Erv46p antisera. Kar2p is shown as a loading control.
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Figure 9: Erv41p and Erv46p depend on each other for their expression. wt, wild-type strain FY834; 46Δ, erv46Δ deletion strain CBY799; 41Δ, erv41Δ deletion strain CBY797; and 41Δ 46Δ, erv41Δ erv46Δ double deletion strain CBY795. Semiintact cells were resolved on a 12.5% polyacrylamide gel and immunoblotted with polyclonal anti-Erv41p and anti-Erv46p antisera. Kar2p is shown as a loading control.

Mentions: Based on the colocalization of Erv41p and Erv46p, the similarity in phenotypes displayed by erv41Δ and erv46Δ in our genetic experiments and the nonadditive effects of the erv41Δ and erv46Δ mutations on ER–Golgi transport assays, we suspected that these proteins act together and possibly form a complex. Therefore, we investigated whether the expression of Erv41p and Erv46p is interdependent by immunoblotting cells containing the erv41Δ, erv46Δ, and erv41Δ erv46Δ alleles with antisera against Erv41p and Erv46p. The erv46Δ strain did not express a detectable level of Erv41p, and the erv41Δ strain exhibited a reduced expression of Erv46p compared with the wild type (Fig. 9). Consistent with these findings, we observed that Erv46p could be expressed at higher levels from a 2μ plasmid than Erv41p (Fig. 3). A strain carrying both ERV41 and ERV46 on 2μ plasmids did not express either of them at levels higher than a strain just overexpressing Erv46p (not shown). Also, the expression level of endogenous Erv41p could be elevated (approximately twofold) if Erv46p was overproduced under control of the GAL1 promoter (compare the total [T] lanes in Fig. 10). These results indicate that the expression of Erv41p is highly dependent on the presence of Erv46p, and to a lesser extent Erv46p expression depends on the presence of Erv41p. One interpretation of these results is that Erv41p and Erv46p are associated in a multisubunit protein complex, and when one of the members of this complex is absent, the other subunit(s) is destabilized.


Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex.

Otte S, Belden WJ, Heidtman M, Liu J, Jensen ON, Barlowe C - J. Cell Biol. (2001)

Erv41p and Erv46p depend on each other for their expression. wt, wild-type strain FY834; 46Δ, erv46Δ deletion strain CBY799; 41Δ, erv41Δ deletion strain CBY797; and 41Δ 46Δ, erv41Δ erv46Δ double deletion strain CBY795. Semiintact cells were resolved on a 12.5% polyacrylamide gel and immunoblotted with polyclonal anti-Erv41p and anti-Erv46p antisera. Kar2p is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195992&req=5

Figure 9: Erv41p and Erv46p depend on each other for their expression. wt, wild-type strain FY834; 46Δ, erv46Δ deletion strain CBY799; 41Δ, erv41Δ deletion strain CBY797; and 41Δ 46Δ, erv41Δ erv46Δ double deletion strain CBY795. Semiintact cells were resolved on a 12.5% polyacrylamide gel and immunoblotted with polyclonal anti-Erv41p and anti-Erv46p antisera. Kar2p is shown as a loading control.
Mentions: Based on the colocalization of Erv41p and Erv46p, the similarity in phenotypes displayed by erv41Δ and erv46Δ in our genetic experiments and the nonadditive effects of the erv41Δ and erv46Δ mutations on ER–Golgi transport assays, we suspected that these proteins act together and possibly form a complex. Therefore, we investigated whether the expression of Erv41p and Erv46p is interdependent by immunoblotting cells containing the erv41Δ, erv46Δ, and erv41Δ erv46Δ alleles with antisera against Erv41p and Erv46p. The erv46Δ strain did not express a detectable level of Erv41p, and the erv41Δ strain exhibited a reduced expression of Erv46p compared with the wild type (Fig. 9). Consistent with these findings, we observed that Erv46p could be expressed at higher levels from a 2μ plasmid than Erv41p (Fig. 3). A strain carrying both ERV41 and ERV46 on 2μ plasmids did not express either of them at levels higher than a strain just overexpressing Erv46p (not shown). Also, the expression level of endogenous Erv41p could be elevated (approximately twofold) if Erv46p was overproduced under control of the GAL1 promoter (compare the total [T] lanes in Fig. 10). These results indicate that the expression of Erv41p is highly dependent on the presence of Erv46p, and to a lesser extent Erv46p expression depends on the presence of Erv41p. One interpretation of these results is that Erv41p and Erv46p are associated in a multisubunit protein complex, and when one of the members of this complex is absent, the other subunit(s) is destabilized.

Bottom Line: The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain.When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains.A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.

Show MeSH
Related in: MedlinePlus