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First published online 31 October 2006
doi: 10.1242/jcs.03250

Journal of Cell Science 119, 4730-4740 (2006)
Published by The Company of Biologists 2006
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Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex

Leah M. Welsh1, Amy Hin Yan Tong2, Charles Boone2, Ole N. Jensen3 and Stefan Otte1,*

1 Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA
2 Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada
3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark


Figure 1
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  		Fig. 1. Synthetic cold-sensitive effects with the erv46 deletion. Wild-type (wt, BY4742), erv46{Delta} (SOY01409), vma12{Delta} (SOY01628), vma12{Delta} erv46{Delta} (SOY01654), vma21{Delta} (SOY01629), vma21{Delta} erv46{Delta} (SOY01655), vma22{Delta} (SOY01630), vma22{Delta} erv46{Delta} (SOY01656), vps1{Delta} (SOY01631), vps1{Delta} erv46{Delta} (SOY01657), vps27{Delta} (SOY01632) and vps27{Delta} erv46{Delta} (SOY01658) cells were grown to saturation in YPD medium and adjusted on an OD600 of 3.0. 5 µl of a 10-fold dilution series were spotted onto YPD plates. One set of plates was incubated at 30°C for 2 days and the other at 16°C for 1 week in order to visualize the synthetic growth phenotype of the double mutants. After this long incubation, the slight cold sensitivity of the erv46{Delta} strain is not visible.

 

Figure 2
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  		Fig. 2. Influence of erv46 and vma21 deletion mutations on {alpha} factor precursor transport and packaging into COPII vesicles. (A) Washed semi-intact wild-type (BY4742), erv46{Delta} (SOY01409), vma21{Delta} (SOY01629) or erv46{Delta} vma21{Delta} (SOY01655) cells containing 35S-labelled gp{alpha}f were incubated without (NA) or with purified COPII subunits, Uso1p and LMA1 (Recon) in the presence of an ATP regeneration system. After 90 minutes at 23°C, outer-chain modified 35S-gp{alpha}f was immunoprecipitated and amounts were plotted relative to 35S-gp{alpha}f precipitated on concanavalin A beads. (B) Washed microsomes prepared from the same strains as in A containing 35S-gp{alpha}f were incubated without (NA) or with purified COPII subunits (COPII) in the presence of an ATP regeneration system. After 20 minutes at 23°C, the relative amounts of gp{alpha}f in the diffusible vesicle fraction were measured. All assays were performed in duplicate and error bars represent the range.

 

Figure 3
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  		Fig. 3. The v-SNARE, Bos1p, is less efficiently sorted into COPII vesicles in a vma21 deletion strain than in the wild type. (A) Microsomes from wild-type (BY4742), erv46{Delta} (SOY01409) or vma21{Delta} (SOY01629) strains were incubated without (–) or with (+) purified COPII subunits in presence of GTP and an ATP regeneration system. Membranes from 10% of the total reaction (T) and vesicle fractions were collected by centrifugation and detected by western blotting with antibodies against various vesicle proteins as well as the ER resident, Sec12p, as a negative control. A line marks the Erv29p band among bands cross-reacting with the polyclonal antiserum. (B,C) Densitometric analysis of three independent experiments performed as in A. Mean packaging efficiencies as a percentage of input were plotted, and error bars represent the standard deviation.

 

Figure 4
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  		Fig. 4. Vesicles from a vma21 deletion strain are fusion competent, and Erv46p is required only on the acceptor membrane. (A) In the first stage of a transport assay, COPII vesicles were generated from wild-type (BY4742), erv46{Delta} (SOY01409) or vma21{Delta} (SOY01629) microsomes containing gp{alpha}f. In the second stage, these vesicles were incubated with washed semi-intact wild-type (BY4742, Recon +wt) or erv46{Delta} (SOY01409, Recon +erv46) cells in presence of Uso1p, LMA1 and the ATP regeneration system. NA, mock reaction with wild-type semi-intact cells but no Uso1p or LMA1; Recon –Acc, control containing Uso1p and LMA1 but no semi-intact cells. Percentage fusion was quantified after precipitation of the outer-chain modified forms of gp{alpha}f. All assays were done in duplicate and error bars represent the range. (B) Overexpression of Bos1p does not suppress the cold-sensitive phenotypes of vma21{Delta} or vma21{Delta} erv46{Delta} strains. Wild-type, single or double deletion strains (SOY163-SOY174) carrying empty vectors or multicopy BOS1 or BET1 overexpression plasmids as indicated were grown in appropriate selective media, spotted on plates and incubated as described in Fig. 1.

 

Figure 5
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  		Fig. 5. Erv41p and Erv46p co-sediment on a sucrose velocity gradient. A post-nuclear supernatant (T) of FY834 cells was solubilized in 1% Triton X-100 (S) and separated on a 5-19% sucrose gradient. Fractions were collected from the top and blotted for Erv41p and Erv46p, as well as for Erv25p, which is present in a 100 kDa complex that serves as a molecular mass marker. Relative amounts of these proteins were determined by densitometry of immunoblots and plotted. The position of the 440 kDa marker ferritin was determined by spectrophotometry.

 

Figure 6
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  		Fig. 6. Glucosidase II is co-immunoprecipitated with the Erv41p-Erv46p complex. (A) Microsomes were prepared from wild-type (FY834) and strains expressing 3HA-Erv46p (CBY770) or 3HA-Erv41p (CBY783) and solubilized with 1% digitonin. Soluble extracts were incubated in the absence (–) or presence (+) of a monoclonal antibody against the HA epitope. Precipitates were resolved on a silver stained gel. Numbers indicate bands representing Erv41p (1), 3HA-Erv41p (2) and Erv46p (3). The band indicated by and asterisk (*) was excised from a Coomassie Blue-stained gel and identified by mass spectrometry as glucosidase II. (B) Microsomes were isolated from wild-type (FY834) and Rot2p-3HA-expressing strains (SOY01117) and solubilized as above. Antibodies against the HA epitope, Erv46p or Erv41 were added as indicated at the top. Proteins eluted from the precipitate (IP), totals (T) and solubilized extracts (S) were resolved on a 12.5% polyacrylamide gel and immunoblotted with 3HA-, Erv46p-, Erv41p- and Erv25p-specific antibodies. T and S each represent 2.6% of input.

 

Figure 7
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  		Fig. 7. Glucosidase II is not packaged into COPII vesicles in vitro and strains lacking a cycling Erv41p-Erv46p complex have a glycoprotein processing defect. (A) Microsomes from a strain expressing Rot2p-3HA (SOY01117) were incubated without (–) or with (+) purified COPII subunits in the presence of GTP and an ATP regeneration system. Membranes from 10% of the total reaction (T) and vesicle fractions were collected by centrifugation, resolved on a polyacrylamide gel and immunoblotted. (B) 35S-labelled {alpha} factor pheromone precursor was translocated into semi-intact cells. Membranes were solubilized and glycoproteins precipitated on concanavalin A Sepharose beads. Bound radiolabelled protein was eluted from the beads, resolved on polyacrylamide gels and detected by phosphorimaging. Fully core glycosylated {alpha} factor pheromone precursor is indicated (gp{alpha}f). The lower bands represent partially glycosylated species. Arrowheads mark the band representing incompletely trimmed gp{alpha}f. Semi-intact cells were prepared from wild-type (wt, FY834), rot2{Delta} (CBY1087), cwh41{Delta} (CBY1086), an erv41{Delta} strain containing empty vector pRS314 (CBY1036), or an erv41{Delta} strain containing the ERV41 gene in vector pRS314 (CBY1037). (C) Same procedure as in B, but semi-intact cells were prepared from an erv41{Delta} strain containing a plasmid which encodes a truncated version of ERV41 lacking C-terminal residues 340-352 (CBY1038), or from erv41{Delta} strains transformed with pRS314 plasmids containing alanine scan ERV41 mutants (CBY1074-1080, CBY1089-1096, CBY1158 and CBY1159, respectively).

 

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© The Company of Biologists Ltd 2006