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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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Pulse–chase analysis of CPY maturation in wild-type and erv14Δ erv46Δ deletion strains. Wild-type (wt, FY833), erv46Δ (46Δ, CBY798), erv14Δ (14Δ, CBY358), and erv14Δ erv46Δ (14Δ 46Δ, CBY822) strains were pulsed for 7 min with 35S-labeled cysteine and methionine and then chased for 10 or 20 min. Labeled CPY was immunoprecipitated from cell extracts, resolved on a 10% polyacrylamide gel, and visualized by autoradiography.
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Figure 7: Pulse–chase analysis of CPY maturation in wild-type and erv14Δ erv46Δ deletion strains. Wild-type (wt, FY833), erv46Δ (46Δ, CBY798), erv14Δ (14Δ, CBY358), and erv14Δ erv46Δ (14Δ 46Δ, CBY822) strains were pulsed for 7 min with 35S-labeled cysteine and methionine and then chased for 10 or 20 min. Labeled CPY was immunoprecipitated from cell extracts, resolved on a 10% polyacrylamide gel, and visualized by autoradiography.

Mentions: Since erv41Δ and erv46Δ strains did not exhibit major defects in secretion, but the erv14Δ erv46Δ strains showed an exacerbated growth phenotype, we examined the transport kinetics in this strain compared with wild-type and the single erv14Δ and erv46Δ strains. A pulse–chase experiment was performed in which cells were grown in minimal media and pulsed for 7 min with [35S]methionine and [35S]cysteine to label newly synthesized proteins. Excess unlabeled cysteine and methionine were added for the chase phase, and the maturation of CPY was followed by immunoprecipitation with an anti-CPY antibody. CPY first appears in the ER as the P1 precursor form of 67 kD and is then modified in the Golgi to yield its P2 form of 69 kD and finally processed in the vacuole to the mature M form of 61 kD (Stevens et al. 1984). As seen in Fig. 7, a slight delay in transport of CPY was observed in an erv14Δ strain (Powers and Barlowe 1998), and, when combined with erv46Δ, no further decrease in the transport rate was detected.


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)

Pulse–chase analysis of CPY maturation in wild-type and erv14Δ erv46Δ deletion strains. Wild-type (wt, FY833), erv46Δ (46Δ, CBY798), erv14Δ (14Δ, CBY358), and erv14Δ erv46Δ (14Δ 46Δ, CBY822) strains were pulsed for 7 min with 35S-labeled cysteine and methionine and then chased for 10 or 20 min. Labeled CPY was immunoprecipitated from cell extracts, resolved on a 10% polyacrylamide gel, and visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Pulse–chase analysis of CPY maturation in wild-type and erv14Δ erv46Δ deletion strains. Wild-type (wt, FY833), erv46Δ (46Δ, CBY798), erv14Δ (14Δ, CBY358), and erv14Δ erv46Δ (14Δ 46Δ, CBY822) strains were pulsed for 7 min with 35S-labeled cysteine and methionine and then chased for 10 or 20 min. Labeled CPY was immunoprecipitated from cell extracts, resolved on a 10% polyacrylamide gel, and visualized by autoradiography.
Mentions: Since erv41Δ and erv46Δ strains did not exhibit major defects in secretion, but the erv14Δ erv46Δ strains showed an exacerbated growth phenotype, we examined the transport kinetics in this strain compared with wild-type and the single erv14Δ and erv46Δ strains. A pulse–chase experiment was performed in which cells were grown in minimal media and pulsed for 7 min with [35S]methionine and [35S]cysteine to label newly synthesized proteins. Excess unlabeled cysteine and methionine were added for the chase phase, and the maturation of CPY was followed by immunoprecipitation with an anti-CPY antibody. CPY first appears in the ER as the P1 precursor form of 67 kD and is then modified in the Golgi to yield its P2 form of 69 kD and finally processed in the vacuole to the mature M form of 61 kD (Stevens et al. 1984). As seen in Fig. 7, a slight delay in transport of CPY was observed in an erv14Δ strain (Powers and Barlowe 1998), and, when combined with erv46Δ, no further decrease in the transport rate was detected.

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