Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex

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.

Figure 2

Selective packaging of Erv proteins into COPII-coated vesicles in vitro. (A) In vitro budding reactions with microsomes prepared from strains expressing tagged versions of Erv proteins: Yif1p-3HA (CBY801), 3HA-Erv29p (CBY950), 3HA-Erv46p (CBY767), and 3HA-Erv41p (CBY782). One tenth of a total reaction (T), budded vesicles isolated after incubation with COPII proteins (+), or a mock reaction without COPII proteins (–) were separated on a 12.5% polyacrylamide gel. Tagged proteins were visualized by immunoblot with an anti-HA antibody, and Sec61p or Sec12p (ER resident proteins) as negative controls and Erv25p (Erv protein) or Bos1p (v-SNARE) as positive controls were detected using polyclonal antisera. (B) In vitro budding reactions with FY834 wild-type microsomes. The same budding protocol as in A was used. Proteins were detected with polyclonal antisera against Sec12p, Kar2p, and Sec61p (ER residents), Bos1p (v-SNARE), Och1p (early Golgi marker), and the Erv proteins Erv46p, Erv41p, Erv25p, Erv14p, Rer1p, and Yip1p.

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.

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).

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.

Figure 9

Erv41p and Erv46p depend on each other for their expression. wt, wild-type strain FY834; 46Δ, erv46Δ deletion strain CBY799; 41Δ, erv41Δ deletion strain CBY797; and 41Δ 46Δ, erv41Δ erv46Δ double deletion strain CBY795. Semiintact cells were resolved on a 12.5% polyacrylamide gel and immunoblotted with polyclonal anti-Erv41p and anti-Erv46p antisera. Kar2p is shown as a loading control.

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.

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.

Figure 7

Pulse–chase analysis of CPY maturation in wild-type and erv14Δ erv46Δ deletion strains. Wild-type (wt, FY833), erv46Δ (46Δ, CBY798), erv14Δ (14Δ, CBY358), and erv14Δ erv46Δ (14Δ 46Δ, CBY822) strains were pulsed for 7 min with ³⁵S-labeled cysteine and methionine and then chased for 10 or 20 min. Labeled CPY was immunoprecipitated from cell extracts, resolved on a 10% polyacrylamide gel, and visualized by autoradiography.

Figure 8

Influences of erv41Δ and erv46Δ mutations on ER–Golgi transport. (A) Vesicle budding and tethering in washed semiintact cells prepared from wild-type (wt, FY834), erv41Δ (41Δ, CBY797), erv46Δ (46Δ, CBY799), and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. The levels of diffusible vesicles in reactions without reconstitution proteins (NA), with COPII proteins (+COPII), and with COPII proteins plus the tethering factor Uso1p (+COPII/Uso1p) are indicated. (B) Overall transport of ³⁵S-labeled gp-α-F to the Golgi complex in the same strains. Semiintact cells were incubated alone (NA) or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon). (C) Overall transport of ³⁵S-labeled gp-α-F factor to the Golgi complex in wild-type (wt, FY834) and erv41Δ erv46Δ (41Δ 46Δ, CBY795) strains. Semiintact cells were incubated alone (NA), with cytosol, or with the reconstitution proteins COPII, Uso1p, and LMA1 (+Recon).

Figure 10

Erv41p is coimmunoprecipitated with HA-tagged Erv46p. Microsomes were prepared from FY833 (− HA-Erv46p) and CBY767 (+ HA-Erv46p) cells grown in YP with 2% galactose and 0.2% glucose and solubilized with buffer containing 2% Triton X-100 (T). The soluble extracts (S) were incubated in absence (− anti-HA ab) or presence (+ anti-HA ab) of the monoclonal anti-HA antibody. Precipitates were resolved on a 12.5% polyacrylamide gel and immunoblotted for Sec23p, Erv46p, Erv41p, and Erv25p.