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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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Identification of Erv proteins. COPII-coated vesicles were synthesized in vitro from ER membranes in the presence of COPII proteins (+), were solubilized in sample buffer, and were resolved on a 15% polyacrylamide gel. As a negative control, a mock reaction without COPII proteins (−) was performed. Proteins were silver stained for this figure. For mass spectroscopy, proteins were stained with colloidal blue, and individual bands were excised.
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Figure 1: Identification of Erv proteins. COPII-coated vesicles were synthesized in vitro from ER membranes in the presence of COPII proteins (+), were solubilized in sample buffer, and were resolved on a 15% polyacrylamide gel. As a negative control, a mock reaction without COPII proteins (−) was performed. Proteins were silver stained for this figure. For mass spectroscopy, proteins were stained with colloidal blue, and individual bands were excised.

Mentions: A protocol to generate ER-derived vesicles in vitro from washed microsomes and purified COPII components (Barlowe et al. 1994) was scaled up to prepare sufficient amounts of material to observe polypeptides on protein-stained polyacrylamide gels (Belden and Barlowe 1996). A set of polypeptides was observed (Fig. 1), and individual bands were subjected to tryptic peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry followed by sequence database searching as described (Shevchenko et al. 1996; Jensen et al. 1999). Several proteins were identified, some that had been previously described and others that had not been characterized. Importantly, three of these proteins (Erv14p, Emp24p, and Erv25p) had been identified in our initial approaches using automated NH2-terminal sequencing (Belden and Barlowe 1996; Powers and Barlowe 1998). Therefore, the isolated ER-derived vesicles are comparable to previous preparations, which suggests that this method should allow for the identification of additional Erv proteins. The SNARE protein Bet1p (Newman and Ferro-Novick 1987) was also identified in this preparation and had been previously detected on ER-derived vesicles by immunoblot analysis and is required for transport between the ER and Golgi (Newman et al. 1992; Cao and Barlowe 2000).


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)

Identification of Erv proteins. COPII-coated vesicles were synthesized in vitro from ER membranes in the presence of COPII proteins (+), were solubilized in sample buffer, and were resolved on a 15% polyacrylamide gel. As a negative control, a mock reaction without COPII proteins (−) was performed. Proteins were silver stained for this figure. For mass spectroscopy, proteins were stained with colloidal blue, and individual bands were excised.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Identification of Erv proteins. COPII-coated vesicles were synthesized in vitro from ER membranes in the presence of COPII proteins (+), were solubilized in sample buffer, and were resolved on a 15% polyacrylamide gel. As a negative control, a mock reaction without COPII proteins (−) was performed. Proteins were silver stained for this figure. For mass spectroscopy, proteins were stained with colloidal blue, and individual bands were excised.
Mentions: A protocol to generate ER-derived vesicles in vitro from washed microsomes and purified COPII components (Barlowe et al. 1994) was scaled up to prepare sufficient amounts of material to observe polypeptides on protein-stained polyacrylamide gels (Belden and Barlowe 1996). A set of polypeptides was observed (Fig. 1), and individual bands were subjected to tryptic peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry followed by sequence database searching as described (Shevchenko et al. 1996; Jensen et al. 1999). Several proteins were identified, some that had been previously described and others that had not been characterized. Importantly, three of these proteins (Erv14p, Emp24p, and Erv25p) had been identified in our initial approaches using automated NH2-terminal sequencing (Belden and Barlowe 1996; Powers and Barlowe 1998). Therefore, the isolated ER-derived vesicles are comparable to previous preparations, which suggests that this method should allow for the identification of additional Erv proteins. The SNARE protein Bet1p (Newman and Ferro-Novick 1987) was also identified in this preparation and had been previously detected on ER-derived vesicles by immunoblot analysis and is required for transport between the ER and Golgi (Newman et al. 1992; Cao and Barlowe 2000).

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