Limits...
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.

Show MeSH

Related in: MedlinePlus

Genetic experiments with erv41Δ and erv46Δ strains. (A) An erv14Δ strain (CBY358) was mated with an erv46Δ strain (CBY799), and spores were dissected on a YPD plate. Spores that germinated and grew slower were shown to carry both deletions. (B) Cold sensitivity of erv14Δ, erv41Δ, and erv46Δ strains. Wild-type (FY834), erv46Δ (CBY799), erv41Δ (CBY797), erv14Δ (CBY356), erv14Δ erv46Δ (CBY823), erv14Δ erv41Δ (CBY825), erv41Δ erv46Δ (CBY795), and erv14Δ erv41Δ erv46Δ (CBY894) strains were grown to saturation in YPD, adjusted to an OD600 of 3.0, and 5 μl of a 10-fold dilution series were spotted onto YPD plates. (C) Effects of erv41Δ and erv46Δ mutations on the ypt1-3 mutation. Wild-type (FY834), ypt1-3 (CBY829), erv46Δ ypt1-3 (CBY843), erv41Δ ypt1-3 (CBY841), erv41Δ erv46Δ ypt1-3 (CBY845), and erv41Δ erv46Δ (CBY795) cells were spotted on YPD plates as in B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195992&req=5

Figure 6: Genetic experiments with erv41Δ and erv46Δ strains. (A) An erv14Δ strain (CBY358) was mated with an erv46Δ strain (CBY799), and spores were dissected on a YPD plate. Spores that germinated and grew slower were shown to carry both deletions. (B) Cold sensitivity of erv14Δ, erv41Δ, and erv46Δ strains. Wild-type (FY834), erv46Δ (CBY799), erv41Δ (CBY797), erv14Δ (CBY356), erv14Δ erv46Δ (CBY823), erv14Δ erv41Δ (CBY825), erv41Δ erv46Δ (CBY795), and erv14Δ erv41Δ erv46Δ (CBY894) strains were grown to saturation in YPD, adjusted to an OD600 of 3.0, and 5 μl of a 10-fold dilution series were spotted onto YPD plates. (C) Effects of erv41Δ and erv46Δ mutations on the ypt1-3 mutation. Wild-type (FY834), ypt1-3 (CBY829), erv46Δ ypt1-3 (CBY843), erv41Δ ypt1-3 (CBY841), erv41Δ erv46Δ ypt1-3 (CBY845), and erv41Δ erv46Δ (CBY795) cells were spotted on YPD plates as in B.

Mentions: The haploid erv41Δ strains (CBY796 and CBY797) grew at rates comparable to wild-type strains at 30°C and 37°C but displayed a reduced growth rate at 16°C (Fig. 6 B). The cellular morphology as determined by light microscopy as well as the mating and sporulation efficiencies of an erv41Δ strain were indistinguishable from the isogenic wild-type strains. Intracellular transport of the secretory proteins CPY and Gas1p was not detectably altered in an erv41Δ strain and the major secretory proteins contained in culture supernatants were also unchanged (data not shown). Therefore, deletion of ERV41 does not appear to interfere with general secretion. We performed a similar set of analyses on erv46Δ strains and observed phenotypes that were identical to erv41Δ strains, notably a reduced growth rate at 16°C. Furthermore, erv41Δ erv46Δ strains did not exhibit any exacerbated phenotypes compared with the single deletions strains (Fig. 6 B).


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)

Genetic experiments with erv41Δ and erv46Δ strains. (A) An erv14Δ strain (CBY358) was mated with an erv46Δ strain (CBY799), and spores were dissected on a YPD plate. Spores that germinated and grew slower were shown to carry both deletions. (B) Cold sensitivity of erv14Δ, erv41Δ, and erv46Δ strains. Wild-type (FY834), erv46Δ (CBY799), erv41Δ (CBY797), erv14Δ (CBY356), erv14Δ erv46Δ (CBY823), erv14Δ erv41Δ (CBY825), erv41Δ erv46Δ (CBY795), and erv14Δ erv41Δ erv46Δ (CBY894) strains were grown to saturation in YPD, adjusted to an OD600 of 3.0, and 5 μl of a 10-fold dilution series were spotted onto YPD plates. (C) Effects of erv41Δ and erv46Δ mutations on the ypt1-3 mutation. Wild-type (FY834), ypt1-3 (CBY829), erv46Δ ypt1-3 (CBY843), erv41Δ ypt1-3 (CBY841), erv41Δ erv46Δ ypt1-3 (CBY845), and erv41Δ erv46Δ (CBY795) cells were spotted on YPD plates as in B.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Genetic experiments with erv41Δ and erv46Δ strains. (A) An erv14Δ strain (CBY358) was mated with an erv46Δ strain (CBY799), and spores were dissected on a YPD plate. Spores that germinated and grew slower were shown to carry both deletions. (B) Cold sensitivity of erv14Δ, erv41Δ, and erv46Δ strains. Wild-type (FY834), erv46Δ (CBY799), erv41Δ (CBY797), erv14Δ (CBY356), erv14Δ erv46Δ (CBY823), erv14Δ erv41Δ (CBY825), erv41Δ erv46Δ (CBY795), and erv14Δ erv41Δ erv46Δ (CBY894) strains were grown to saturation in YPD, adjusted to an OD600 of 3.0, and 5 μl of a 10-fold dilution series were spotted onto YPD plates. (C) Effects of erv41Δ and erv46Δ mutations on the ypt1-3 mutation. Wild-type (FY834), ypt1-3 (CBY829), erv46Δ ypt1-3 (CBY843), erv41Δ ypt1-3 (CBY841), erv41Δ erv46Δ ypt1-3 (CBY845), and erv41Δ erv46Δ (CBY795) cells were spotted on YPD plates as in B.
Mentions: The haploid erv41Δ strains (CBY796 and CBY797) grew at rates comparable to wild-type strains at 30°C and 37°C but displayed a reduced growth rate at 16°C (Fig. 6 B). The cellular morphology as determined by light microscopy as well as the mating and sporulation efficiencies of an erv41Δ strain were indistinguishable from the isogenic wild-type strains. Intracellular transport of the secretory proteins CPY and Gas1p was not detectably altered in an erv41Δ strain and the major secretory proteins contained in culture supernatants were also unchanged (data not shown). Therefore, deletion of ERV41 does not appear to interfere with general secretion. We performed a similar set of analyses on erv46Δ strains and observed phenotypes that were identical to erv41Δ strains, notably a reduced growth rate at 16°C. Furthermore, erv41Δ erv46Δ strains did not exhibit any exacerbated phenotypes compared with the single deletions strains (Fig. 6 B).

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