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First published online March 22, 2006
doi: 10.1242/10.1242/jcs.02841

Journal of Cell Science 119, 1406-1415 (2006)
Published by The Company of Biologists 2006
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Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast

Hideki Nakanishi1, Masayo Morishita2, Cindi L. Schwartz3, Alison Coluccio1, JoAnne Engebrecht2 and Aaron M. Neiman1,*

1 Department of Biochemistry and Cell Biology, SUNY Stony Brook, Stony Brook, NY 4-5215, USA
2 Section of Molecular and Cellular Biology, UC Davis, Davis, CA 95616, USA
3 Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, Boulder, CO 80309, USA


Figure 1
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  		Fig. 1. Vesicles docked to the SPB accumulate in spo14 and sso1 mutants. (A) TEM image of a spo14 {Delta}/spo14 {Delta} (HI51) cell in meiosis II, SPB region is boxed. (B) Higher magnification view of the boxed region in A showing vesicles decorating the SPB. (C) A 1 nm Z-slice of a tomogram of a spo14 SPB (for the complete tomogram see Movie 1 in supplementary material). (D) Outlines of vesicles, the outer plaque of the SPB and the central plaque of the SPB shown in purple, green, and light blue, respectively, in the same Z-slice. (E,F) Higher-magnification images from the tomogram of individual vesicles docked at the SPB. Arrows indicate sites of apparent contact between the outer plaque and the vesicular membrane. (G,H) Two views of a three-dimensional reconstruction of a spo14 outer plaque modeled from the tomogram (see Movie 2 in supplementary material). Vesicles are in purple, the outer plaque surface is outlined in green, and the central plaque surface is outlined in light blue as in D. (I) TEM image of a sso1 cell in meiosis II, SPB region is boxed. (J) Higher magnification view of the boxed region in I, showing vesicles decorating the SPB. Bars, 500 nm (A,I); 100 nm (B,C,D,J); 40 nm (E,F).

 

Figure 2
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  		Fig. 2. Prospore membrane proteins accumulate at the SPB in spo14 and sso1 mutants. Wildtype (HI54), sso1 {Delta}/sso1 {Delta} (HI58) and spo14 {Delta}/spo14 {Delta} (HI51) cells expressing the prospore membrane markers GFP-Spo14K1098H (from pRS424-GFP-SPO14K1098H) or Dtr1-GFP (from pRS424-DTR1-GFP) were sporulated and examined by fluorescence microscopy. DNA was visualized using DAPI and SPBs were visualized using an RFP fusion to Mpc54p. Bars, 1 {Delta}m.

 

Figure 3
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  		Fig. 3. Inactivation of SEC14 blocks vesicle accumulation at the SPB in spo14 mutants. sec14-1/sec14-1 (Y5671) and sec14-1/sec14-1 spo14 {Delta}/spo14 {Delta} (Y5688) cells carry pRS424-Dtr1p-GFP were sporulated at either the permissive temperature (24°C) or the nonpermissive temperature (35°C) and examined by fluorescence microscopy. SPBs were visualized using an RFP fusion to Mpc54p. Bars, 5 {Delta}m.

 

Figure 4
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  		Fig. 4. Sorbitol density gradient fractionation of sporulating wildtype, spo14 {Delta} and sso1 {Delta} cells. (A) Immunoblots of fractionated extracts from wild-type (AN120), spo14 (HI6), and sso1 (HI3) cells expressing Dtr1-GFP. The 14,000 g supernatant (S14), pellet (P14), 100,000 g supernatant (S100), pellet (P100) and 20-60% sorbitol gradient fractions (1-22) were probed with anti-GFP to detect Dtr1p-GFP, anti-Gas1p and anti-Dpm1p, or anti-Vps10p (details in Materials and Methods). (B) Quantification of the pixel intensity of each band was performed using AlphaEase FC4.0 imager software. bullet, WT; {blacksquare}, spo14; {blacktriangleup}, sso1.

 

Figure 5
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  		Fig. 5. The membrane-binding domain of Spo20p does not localize to prospore membrane precursor vesicles in the spo14 mutant. Wild-type (HI54), sso1 {Delta}/sso1 {Delta} (HI58) and spo14 {Delta}/spo14 {Delta} (HI51) cells expressing GFP-Spo20p51-91 (from pRS426-G20) were sporulated and examined by fluorescence microscopy. DNA was visualized using DAPI and SPBs were visualized using an RFP fusion to Mpc54p. Bars, 1 {Delta}m.

 

Figure 6
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  		Fig. 6. Prospore membrane proteins accumulate at the SPB in sec1 and spo20 sec9 but not mpc54 mutants. sec1ts/sec1ts (AN127), sec9ts/sec9ts, spo20 {Delta}/spo20 {Delta} (AN211) mpc54 {Delta}/mpc54 {Delta} (NY541) cells expressing GFP-Spo20p51-91 (from pRS426-G20 or pRS424-G20) were sporulated and examined by fluorescence microscopy. DNA was visualized using DAPI. Bars, 1 {Delta}m.

 

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© The Company of Biologists Ltd 2006