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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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Sucrose gradient fractionation of Erv41p, Erv46p, Och1p, and Rer1p. An FY834 whole cell lysate was separated on an 18–60% sucrose density gradient, and fractions were collected, starting with fraction 1 at the top. (A) Relative levels of Emp47p (Golgi marker), Kar2p (ER marker), and plasma membrane marker (PMA) in each fraction were quantified by densitometry of immunoblots. (B) Relative levels of Och1p, Erv46p, Erv41p, and Rer1p as determined by densitometry of the immunoblots shown in C.
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Figure 5: Sucrose gradient fractionation of Erv41p, Erv46p, Och1p, and Rer1p. An FY834 whole cell lysate was separated on an 18–60% sucrose density gradient, and fractions were collected, starting with fraction 1 at the top. (A) Relative levels of Emp47p (Golgi marker), Kar2p (ER marker), and plasma membrane marker (PMA) in each fraction were quantified by densitometry of immunoblots. (B) Relative levels of Och1p, Erv46p, Erv41p, and Rer1p as determined by densitometry of the immunoblots shown in C.

Mentions: The abundant Erv proteins are hypothesized to cycle between the ER and Golgi (Belden and Barlowe 1996; Powers and Barlowe 1998) because they localize to both of these membrane compartments, and, in contrast to abundant secretory proteins, their levels are not diminished by cycloheximide treatment (Yeung et al. 1995). Therefore, we examined the subcellular localization of Erv41p and Erv46p by resolution of membrane organelles on sucrose gradients (Fig. 5). Erv41p and Erv46p sedimented in two peaks, one that coincided with the Golgi marker Emp47p and the other with the ER marker Kar2p. This subcellular localization pattern was similar to that of Erv14p (Powers and Barlowe 1998) and Erv25p (Belden and Barlowe 1996). Rer1p has been shown to be predominantly localized to the Golgi (Sato et al. 1995) and to recycle between the Golgi and ER (Boehm et al. 1997). Consequently, we find that the majority of Rer1p cosedimented with Emp47p, and a second smaller peak cosedimented with Kar2p. In contrast, Och1p was localized almost exclusively to the Golgi. The localization of the epitope-tagged Erv46p was also examined by indirect immunofluorescence using the anti-HA antibody (data not shown). We observed a perinuclear staining pattern that partially overlapped with the ER marker Kar2p and was similar to that described for Erv14p (Powers and Barlowe 1998). We conclude that Erv41p and Erv46p localize to the early secretory pathway.


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)

Sucrose gradient fractionation of Erv41p, Erv46p, Och1p, and Rer1p. An FY834 whole cell lysate was separated on an 18–60% sucrose density gradient, and fractions were collected, starting with fraction 1 at the top. (A) Relative levels of Emp47p (Golgi marker), Kar2p (ER marker), and plasma membrane marker (PMA) in each fraction were quantified by densitometry of immunoblots. (B) Relative levels of Och1p, Erv46p, Erv41p, and Rer1p as determined by densitometry of the immunoblots shown in C.
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Related In: Results  -  Collection

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

Figure 5: Sucrose gradient fractionation of Erv41p, Erv46p, Och1p, and Rer1p. An FY834 whole cell lysate was separated on an 18–60% sucrose density gradient, and fractions were collected, starting with fraction 1 at the top. (A) Relative levels of Emp47p (Golgi marker), Kar2p (ER marker), and plasma membrane marker (PMA) in each fraction were quantified by densitometry of immunoblots. (B) Relative levels of Och1p, Erv46p, Erv41p, and Rer1p as determined by densitometry of the immunoblots shown in C.
Mentions: The abundant Erv proteins are hypothesized to cycle between the ER and Golgi (Belden and Barlowe 1996; Powers and Barlowe 1998) because they localize to both of these membrane compartments, and, in contrast to abundant secretory proteins, their levels are not diminished by cycloheximide treatment (Yeung et al. 1995). Therefore, we examined the subcellular localization of Erv41p and Erv46p by resolution of membrane organelles on sucrose gradients (Fig. 5). Erv41p and Erv46p sedimented in two peaks, one that coincided with the Golgi marker Emp47p and the other with the ER marker Kar2p. This subcellular localization pattern was similar to that of Erv14p (Powers and Barlowe 1998) and Erv25p (Belden and Barlowe 1996). Rer1p has been shown to be predominantly localized to the Golgi (Sato et al. 1995) and to recycle between the Golgi and ER (Boehm et al. 1997). Consequently, we find that the majority of Rer1p cosedimented with Emp47p, and a second smaller peak cosedimented with Kar2p. In contrast, Och1p was localized almost exclusively to the Golgi. The localization of the epitope-tagged Erv46p was also examined by indirect immunofluorescence using the anti-HA antibody (data not shown). We observed a perinuclear staining pattern that partially overlapped with the ER marker Kar2p and was similar to that described for Erv14p (Powers and Barlowe 1998). We conclude that Erv41p and Erv46p localize to the early secretory pathway.

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