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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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Erv46p and Erv41p are integral membrane proteins. (A) Hydrophilicity plots according to Kyte and Doolittle 1982 with a window size of 7. The schematic representations of the polypeptide chains indicate the positions of the putative transmembrane domains (TM1 and TM2) predicted by the HMMTOP program (Tusnady and Simon 1998), the region containing conserved cysteine residues (Cys) and the KKXX motif (black dot). (B) Semiintact FY834 cells were treated with buffer (Materials and Methods), buffer containing 1% Triton X-100 (TX100), or 0.1 M Na2CO3 (pH 11) and centrifuged at 100,000 g. Totals before centrifugation (T), supernatant (S), and pellet (P) fractions were analyzed on a 12.5% polyacrylamide gel and immunoblotted for Sec23p (peripheral membrane protein), Erv46p, Erv41p, and Bos1p (integral membrane protein).
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Figure 4: Erv46p and Erv41p are integral membrane proteins. (A) Hydrophilicity plots according to Kyte and Doolittle 1982 with a window size of 7. The schematic representations of the polypeptide chains indicate the positions of the putative transmembrane domains (TM1 and TM2) predicted by the HMMTOP program (Tusnady and Simon 1998), the region containing conserved cysteine residues (Cys) and the KKXX motif (black dot). (B) Semiintact FY834 cells were treated with buffer (Materials and Methods), buffer containing 1% Triton X-100 (TX100), or 0.1 M Na2CO3 (pH 11) and centrifuged at 100,000 g. Totals before centrifugation (T), supernatant (S), and pellet (P) fractions were analyzed on a 12.5% polyacrylamide gel and immunoblotted for Sec23p (peripheral membrane protein), Erv46p, Erv41p, and Bos1p (integral membrane protein).

Mentions: Both Erv41p and Erv46p contain two segments of sufficient length and hydrophobicity to form transmembrane domains, and the short NH2- and COOH-terminal regions of both proteins are predicted to be located on the cytoplasmic side with a large lumenal domain separating the two transmembrane regions. The hydrophilicity plots of both proteins are superimposable (Fig. 4 A), and a similar topology is predicted for all of the known homologues. There is no evidence for a cleavable signal sequence. To confirm that Erv41p and Erv46p are indeed integral membrane proteins, we examined their fractionation behavior under conditions that release lumenal and peripherally bound membrane proteins (carbonate buffer, pH 11.0) or solubilize integral membrane proteins (1% Triton X-100). The fractionation profile for Erv41p and Erv46p was the same as for the integral membrane protein Bos1p, as they could only be solubilized by detergent and not by carbonate treatment (Fig. 4 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)

Erv46p and Erv41p are integral membrane proteins. (A) Hydrophilicity plots according to Kyte and Doolittle 1982 with a window size of 7. The schematic representations of the polypeptide chains indicate the positions of the putative transmembrane domains (TM1 and TM2) predicted by the HMMTOP program (Tusnady and Simon 1998), the region containing conserved cysteine residues (Cys) and the KKXX motif (black dot). (B) Semiintact FY834 cells were treated with buffer (Materials and Methods), buffer containing 1% Triton X-100 (TX100), or 0.1 M Na2CO3 (pH 11) and centrifuged at 100,000 g. Totals before centrifugation (T), supernatant (S), and pellet (P) fractions were analyzed on a 12.5% polyacrylamide gel and immunoblotted for Sec23p (peripheral membrane protein), Erv46p, Erv41p, and Bos1p (integral membrane protein).
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Figure 4: Erv46p and Erv41p are integral membrane proteins. (A) Hydrophilicity plots according to Kyte and Doolittle 1982 with a window size of 7. The schematic representations of the polypeptide chains indicate the positions of the putative transmembrane domains (TM1 and TM2) predicted by the HMMTOP program (Tusnady and Simon 1998), the region containing conserved cysteine residues (Cys) and the KKXX motif (black dot). (B) Semiintact FY834 cells were treated with buffer (Materials and Methods), buffer containing 1% Triton X-100 (TX100), or 0.1 M Na2CO3 (pH 11) and centrifuged at 100,000 g. Totals before centrifugation (T), supernatant (S), and pellet (P) fractions were analyzed on a 12.5% polyacrylamide gel and immunoblotted for Sec23p (peripheral membrane protein), Erv46p, Erv41p, and Bos1p (integral membrane protein).
Mentions: Both Erv41p and Erv46p contain two segments of sufficient length and hydrophobicity to form transmembrane domains, and the short NH2- and COOH-terminal regions of both proteins are predicted to be located on the cytoplasmic side with a large lumenal domain separating the two transmembrane regions. The hydrophilicity plots of both proteins are superimposable (Fig. 4 A), and a similar topology is predicted for all of the known homologues. There is no evidence for a cleavable signal sequence. To confirm that Erv41p and Erv46p are indeed integral membrane proteins, we examined their fractionation behavior under conditions that release lumenal and peripherally bound membrane proteins (carbonate buffer, pH 11.0) or solubilize integral membrane proteins (1% Triton X-100). The fractionation profile for Erv41p and Erv46p was the same as for the integral membrane protein Bos1p, as they could only be solubilized by detergent and not by carbonate treatment (Fig. 4 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.

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Related in: MedlinePlus