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First published online 13 February 2007
doi: 10.1242/jcs.03405

Journal of Cell Science 120, 908-916 (2007)
Published by The Company of Biologists 2007
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Erv14 family cargo receptors are necessary for ER exit during sporulation in Saccharomyces cerevisiae

Hideki Nakanishi*, Yasuyuki Suda* and Aaron M. Neiman{ddagger}

Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, SUNY Stony Brook, Stony Brook, NY 11794-5215, USA


Figure 1
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  		Fig. 1. Prospore membrane formation mutants. Wild-type (AN120), rcy1{Delta}/rcy1{Delta} (31221), sec22{Delta} /sec22{Delta} (35177), vps13{Delta} /vps13{Delta} (HI29), erv14{Delta} /erv14{Delta} (HI26) and sma2{Delta} /sma2{Delta} (HI44) cells were transformed with pRS426-G20 and the localization of the prospore membrane marker GFP-Spo2051-91 was observed in late Meiosis II cells. sec22{Delta}/sec22{Delta} and rcy1{Delta}/rcy1{Delta} mutants are BY4743 background, the others are in the SK-1 background. The corresponding DNA images are shown (DAPI). Bars, 1 µm.

 

Figure 2
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  		Fig. 2. Overexpression of SMA2 partially suppresses the sporulation defect of an erv14{Delta} mutant. (A) Sma2p-GFP localizes to the prospore membrane. Sma2p-GFP expressed from pRS424-SPO20pr-SMA2-GFP was observed in a sporulating wild type cell (AN120). DNA was stained with DAPI (DAPI). Bar, 1 µm. (B) Overexpression of SMA2 restores sporulation to the erv14{Delta} mutant. erv14{Delta}/erv14{Delta} (H126) cells harboring pRS314-ERV14HA, pRS424-SPO20pr-SMA2 or empty vector, were sporulated and 3x105 cells were spotted onto a YPD plate, exposed to ether vapor and then incubated at 30°C.

 

Figure 3
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  		Fig. 3. Sma2p-GFP accumulates in the ER in vegetative and sporulating erv14{Delta} cells. Wild-type (AN120) and erv14{Delta}/erv14{Delta} (HI26) cells were transformed with pRS424-SSO1pr-Sma2-GFP and YEp352-GAPII-KmRFP (Kar2-RFP), or with pRS424-SPO20pr-Sma2-GFP and pRS426-SPO20pr-SEC61-mRFP, and the localization of Sma2-GFP and the RFP-tagged ER markers was observed in vegetative and sporulating cells. The corresponding DNA images are shown (DAPI). Bar, 1 µm.

 

Figure 4
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  		Fig. 4. Overexpression of ERV15 suppresses the sporulation defect of an erv14{Delta} mutant. Wild-type (AN120) cells harboring the empty vector pRS424, or erv14{Delta}/erv14{Delta} (H126) cells harboring pRS314-ERV14HA, pRS424-ERV15 or empty vector were sporulated and 3x105 cells were spotted onto a YPD plate, exposed to ether vapor and incubated at 30°C.

 

Figure 5
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  		Fig. 5. Prospore membrane formation is blocked in erv14{Delta} erv15{Delta} double mutants. Wild-type (AN120) and erv14{Delta}/erv14{Delta} erv15{Delta}/erv15{Delta} (YS232) cells were transformed with pRS426-G20 and pRS304-MPC54-RFP, and the localization of the prospore membrane marker GFP-Spo20p51-91 and the SPB marker Mpc54-RFP were observed in sporulating cells. Bars, 1 µm.

 

Figure 6
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  		Fig. 6. GFP-Sso1p accumulates in the ER in sporulating erv14{Delta} erv15{Delta} double mutants. The localization of GFP-Sso1p was examined in vegetative growth and sporulation in wild-type (AN120), erv14{Delta} (HI26), erv15{Delta} (YS229) and erv14{Delta} erv15{Delta} (YS232) strains. For analysis of vegetative cells, GFP-Sso1p was expressed under the control of TEF2 promoter from pRS424-TEF2pr-GFP-SSO1+3' and the ER was labeled with Kar2-RFP expressed from YEpGAPII-KmRFP. In sporulating cells, GFP-Sso1p and Sec61p-RFP were expressed from sporulation-specific promoters in plasmids pRS424-SPO20pr-GFP-SSO1+3' and pRS426-SPO20pr-SEC61-mRFP, respectively. In the bottom panel, localization of Sso1p to the prospore membrane in erv14{Delta} (HI26) cells was examined using the prospore membrane marker RFP-Spo2051-91. The corresponding DNA images are shown (DAPI). Bar, 1 µm.

 

Figure 7
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  		Fig. 7. Multiple proteins require ERV genes for localization to the prospore membrane during sporulation. Localization of Pma1p, Sso1p, Dtr1p and Sma2p during vegetative growth and sporulation in wild-type (AN120), erv14{Delta} (HI26), erv15{Delta} (YS229) and erv14{Delta} erv15{Delta} (YS232) strains was quantified using GFP fusions and ER markers as in Fig. 6. All the GFP fusions were expressed vegetatively under control of the TEF2 promoter and during sporulation under control of the SPO20 promoter except Dtr1p-GFP, which was expressed from its native promoter. The ER markers used were Kar2-RFP in vegetative cells and Sec61p-RFP in sporulating cells. For each strain at least 100 cells were examined and the percentage of cells showing localization of the GFP fusion to the plasma membrane during vegetative growth (open bars) or prospore membrane during sporulation (black bars) are shown. Asterisks in the Sma2-GFP graph indicate that the Sma2-GFP was mainly found diffusely throughout the cytoplasm in wild-type and erv15{Delta} cells during vegetative growth.

 

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© The Company of Biologists Ltd 2007