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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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Influences of erv41Δ and erv46Δ mutations on ER–Golgi transport. (A) Vesicle budding and tethering in washed semiintact cells prepared from wild-type (wt, FY834), erv41Δ (41Δ, CBY797), erv46Δ (46Δ, CBY799), and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. The levels of diffusible vesicles in reactions without reconstitution proteins (NA), with COPII proteins (+COPII), and with COPII proteins plus the tethering factor Uso1p (+COPII/Uso1p) are indicated. (B) Overall transport of 35S-labeled gp-α-F to the Golgi complex in the same strains. Semiintact cells were incubated alone (NA) or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon). (C) Overall transport of 35S-labeled gp-α-F factor to the Golgi complex in wild-type (wt, FY834) and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. Semiintact cells were incubated alone (NA), with cytosol, or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon).
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Figure 8: Influences of erv41Δ and erv46Δ mutations on ER–Golgi transport. (A) Vesicle budding and tethering in washed semiintact cells prepared from wild-type (wt, FY834), erv41Δ (41Δ, CBY797), erv46Δ (46Δ, CBY799), and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. The levels of diffusible vesicles in reactions without reconstitution proteins (NA), with COPII proteins (+COPII), and with COPII proteins plus the tethering factor Uso1p (+COPII/Uso1p) are indicated. (B) Overall transport of 35S-labeled gp-α-F to the Golgi complex in the same strains. Semiintact cells were incubated alone (NA) or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon). (C) Overall transport of 35S-labeled gp-α-F factor to the Golgi complex in wild-type (wt, FY834) and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. Semiintact cells were incubated alone (NA), with cytosol, or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon).

Mentions: To further investigate a possible defect in ER to Golgi transport, we used an in vitro transport assay that permitted us to differentiate between the budding, tethering, and fusion stages (Barlowe 1997; Cao et al. 1998). We have found this assay to be a more sensitive method to measure transport between the ER and Golgi. In some instances, mutant strains that display normal transport kinetics in pulse–chase experiments show defects in distinct stages of cell-free transport (Conchon et al. 1999). Washed semiintact cells containing 35S-labeled gp-α-F bud 35S-labeled gp-α-F–containing vesicles in the presence of COPII proteins. Packaged 35S-labeled gp-α-F in vesicles can be quantified by precipitation with concanavalin A–Sepharose, allowing us to assay budding efficiencies. The vesicle tethering stage may be monitored as the decrease in diffusible COPII vesicles upon addition of the tethering protein Uso1p. Lastly, reconstituted transport to the Golgi complex can be measured after addition of COPII, Uso1p, and LMA1 to semiintact cells by precipitation of Golgi-modified forms of 35S-labeled gp-α-F with α1,6-mannose–specific antiserum. As seen in Fig. 8 A, the budding and tethering stages of transport were not impaired in mutant strains compared with a wild-type strain. However, there was a modest but significant decrease in transport to the Golgi complex in the erv41Δ, erv46Δ, and the double erv41Δ erv46Δ membranes (Fig. 8 B). Notably, the effects of the single deletions were neither additive nor cooperative when combined in the double mutant strain. Together, these results suggest that the transport defect occurred during the fusion stage of this assay. Also, we examined transport efficiencies in the presence of a crude cytosol to determine if the erv41Δ erv46Δ membranes required additional factors not provided by purified reconstitution proteins. As shown in Fig. 8 C, a similar transport defect was observed for reactions using crude cytosol or purified proteins to drive transport.


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)

Influences of erv41Δ and erv46Δ mutations on ER–Golgi transport. (A) Vesicle budding and tethering in washed semiintact cells prepared from wild-type (wt, FY834), erv41Δ (41Δ, CBY797), erv46Δ (46Δ, CBY799), and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. The levels of diffusible vesicles in reactions without reconstitution proteins (NA), with COPII proteins (+COPII), and with COPII proteins plus the tethering factor Uso1p (+COPII/Uso1p) are indicated. (B) Overall transport of 35S-labeled gp-α-F to the Golgi complex in the same strains. Semiintact cells were incubated alone (NA) or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon). (C) Overall transport of 35S-labeled gp-α-F factor to the Golgi complex in wild-type (wt, FY834) and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. Semiintact cells were incubated alone (NA), with cytosol, or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon).
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Figure 8: Influences of erv41Δ and erv46Δ mutations on ER–Golgi transport. (A) Vesicle budding and tethering in washed semiintact cells prepared from wild-type (wt, FY834), erv41Δ (41Δ, CBY797), erv46Δ (46Δ, CBY799), and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. The levels of diffusible vesicles in reactions without reconstitution proteins (NA), with COPII proteins (+COPII), and with COPII proteins plus the tethering factor Uso1p (+COPII/Uso1p) are indicated. (B) Overall transport of 35S-labeled gp-α-F to the Golgi complex in the same strains. Semiintact cells were incubated alone (NA) or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon). (C) Overall transport of 35S-labeled gp-α-F factor to the Golgi complex in wild-type (wt, FY834) and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. Semiintact cells were incubated alone (NA), with cytosol, or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon).
Mentions: To further investigate a possible defect in ER to Golgi transport, we used an in vitro transport assay that permitted us to differentiate between the budding, tethering, and fusion stages (Barlowe 1997; Cao et al. 1998). We have found this assay to be a more sensitive method to measure transport between the ER and Golgi. In some instances, mutant strains that display normal transport kinetics in pulse–chase experiments show defects in distinct stages of cell-free transport (Conchon et al. 1999). Washed semiintact cells containing 35S-labeled gp-α-F bud 35S-labeled gp-α-F–containing vesicles in the presence of COPII proteins. Packaged 35S-labeled gp-α-F in vesicles can be quantified by precipitation with concanavalin A–Sepharose, allowing us to assay budding efficiencies. The vesicle tethering stage may be monitored as the decrease in diffusible COPII vesicles upon addition of the tethering protein Uso1p. Lastly, reconstituted transport to the Golgi complex can be measured after addition of COPII, Uso1p, and LMA1 to semiintact cells by precipitation of Golgi-modified forms of 35S-labeled gp-α-F with α1,6-mannose–specific antiserum. As seen in Fig. 8 A, the budding and tethering stages of transport were not impaired in mutant strains compared with a wild-type strain. However, there was a modest but significant decrease in transport to the Golgi complex in the erv41Δ, erv46Δ, and the double erv41Δ erv46Δ membranes (Fig. 8 B). Notably, the effects of the single deletions were neither additive nor cooperative when combined in the double mutant strain. Together, these results suggest that the transport defect occurred during the fusion stage of this assay. Also, we examined transport efficiencies in the presence of a crude cytosol to determine if the erv41Δ erv46Δ membranes required additional factors not provided by purified reconstitution proteins. As shown in Fig. 8 C, a similar transport defect was observed for reactions using crude cytosol or purified proteins to drive transport.

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