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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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Immunoblot analysis of wild-type, erv41Δ, and erv46Δ deletion and overproducing strains. FY834 wild-type (wt), erv46Δ (46Δ, CBY799), and erv41Δ (41Δ, CBY797) strains were grown in YPD medium. Heterozygous diploid strains expressing NH2 terminally 3HA-tagged Erv46p (46/HA46, CBY767) or Erv41p (41/HA41, CBY782) were grown in YP with 1.5% galactose and 0.5% glucose. erv14Δ erv46Δ deletion strains carrying pRS426-ERV46 plasmid (2μ 46, CBY836) or pRS424-ERV41 plasmid (2μ 41, CBY847) were grown in minimal media lacking uracil or tryptophan, respectively. Membrane fractions were resolved on a 12.5% polyacrylamide gel and immunoblotted with an anti-Kar2p antiserum as loading control and with polyclonal antisera raised against Erv46p or Erv41p.
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Figure 3: Immunoblot analysis of wild-type, erv41Δ, and erv46Δ deletion and overproducing strains. FY834 wild-type (wt), erv46Δ (46Δ, CBY799), and erv41Δ (41Δ, CBY797) strains were grown in YPD medium. Heterozygous diploid strains expressing NH2 terminally 3HA-tagged Erv46p (46/HA46, CBY767) or Erv41p (41/HA41, CBY782) were grown in YP with 1.5% galactose and 0.5% glucose. erv14Δ erv46Δ deletion strains carrying pRS426-ERV46 plasmid (2μ 46, CBY836) or pRS424-ERV41 plasmid (2μ 41, CBY847) were grown in minimal media lacking uracil or tryptophan, respectively. Membrane fractions were resolved on a 12.5% polyacrylamide gel and immunoblotted with an anti-Kar2p antiserum as loading control and with polyclonal antisera raised against Erv46p or Erv41p.

Mentions: ERV41 as well as ERV46 were amplified from genomic DNA and inserted into the multicopy shuttle vectors pRS424 and pRS426, respectively, to yield plasmids pRS424-ERV41 and pRS426-ERV46. Polyclonal antisera were raised against recombinant forms of Erv41p and Erv46p and then tested on wild-type, deletion, and overproducing strains to confirm their specificity (Fig. 3). No immunoreactive species were detected in the deletion strains, whereas the heterozygous diploid strains carrying one wild-type and one tagged copy of the respective genes expressed two immunoreactive species at the expected size. This result also demonstrated that expression levels from the GAL1 promoter were comparable to wild-type expression levels at a concentration of 1.5% galactose. Strains harboring ERV41 or ERV46 on 2μ based multicopy plasmids expressed slightly higher levels of Erv41p or Erv46p than wild-type strains.


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)

Immunoblot analysis of wild-type, erv41Δ, and erv46Δ deletion and overproducing strains. FY834 wild-type (wt), erv46Δ (46Δ, CBY799), and erv41Δ (41Δ, CBY797) strains were grown in YPD medium. Heterozygous diploid strains expressing NH2 terminally 3HA-tagged Erv46p (46/HA46, CBY767) or Erv41p (41/HA41, CBY782) were grown in YP with 1.5% galactose and 0.5% glucose. erv14Δ erv46Δ deletion strains carrying pRS426-ERV46 plasmid (2μ 46, CBY836) or pRS424-ERV41 plasmid (2μ 41, CBY847) were grown in minimal media lacking uracil or tryptophan, respectively. Membrane fractions were resolved on a 12.5% polyacrylamide gel and immunoblotted with an anti-Kar2p antiserum as loading control and with polyclonal antisera raised against Erv46p or Erv41p.
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Related In: Results  -  Collection

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

Figure 3: Immunoblot analysis of wild-type, erv41Δ, and erv46Δ deletion and overproducing strains. FY834 wild-type (wt), erv46Δ (46Δ, CBY799), and erv41Δ (41Δ, CBY797) strains were grown in YPD medium. Heterozygous diploid strains expressing NH2 terminally 3HA-tagged Erv46p (46/HA46, CBY767) or Erv41p (41/HA41, CBY782) were grown in YP with 1.5% galactose and 0.5% glucose. erv14Δ erv46Δ deletion strains carrying pRS426-ERV46 plasmid (2μ 46, CBY836) or pRS424-ERV41 plasmid (2μ 41, CBY847) were grown in minimal media lacking uracil or tryptophan, respectively. Membrane fractions were resolved on a 12.5% polyacrylamide gel and immunoblotted with an anti-Kar2p antiserum as loading control and with polyclonal antisera raised against Erv46p or Erv41p.
Mentions: ERV41 as well as ERV46 were amplified from genomic DNA and inserted into the multicopy shuttle vectors pRS424 and pRS426, respectively, to yield plasmids pRS424-ERV41 and pRS426-ERV46. Polyclonal antisera were raised against recombinant forms of Erv41p and Erv46p and then tested on wild-type, deletion, and overproducing strains to confirm their specificity (Fig. 3). No immunoreactive species were detected in the deletion strains, whereas the heterozygous diploid strains carrying one wild-type and one tagged copy of the respective genes expressed two immunoreactive species at the expected size. This result also demonstrated that expression levels from the GAL1 promoter were comparable to wild-type expression levels at a concentration of 1.5% galactose. Strains harboring ERV41 or ERV46 on 2μ based multicopy plasmids expressed slightly higher levels of Erv41p or Erv46p than wild-type strains.

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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Related in: MedlinePlus