Volume 8 issue 9 September 2007
Snc1p v-SNARE Transport to the Prospore Membrane During Yeast Sporulation is Dependent on Endosomal Retrieval Pathways
M Morishita, R Mendonsa, J Wright and JA Engebrecht

Supplementary Figure 1: GARP mutants are cold sensitive for growth. The growth phenotype was observed in the GARP mutants a) and the retromer mutants and retromer GARP double mutant b).Wild-type (AN120), vps51Δ (Y5975), vps52Δ Y6092), vps53Δ (Y6377), vps54Δ (Y6287), vps54Δ vps17Δ(Y6723) vps26Δ Y6001) vps35Δ Y5919) and vps17Δ (Y6422) cells were streaked on YPD + 10 mM adenine solid medium for single colonies and incubated at indicated temperatures for 2-7 days. The GARP mutants, vps52Δ, vps53Δ, vps54Δ, and vps54Δ vps17Δ grew slowly compared to WT at 15/16 °C, while the retromer single mutants, vps26Δ, vps35Δ, and vps17Δ grew similar to WT at all temperatures.

Figure 1 (.gif)

Supplementary Figure 2: The actin cytoskeleton is abnormal in vps54Δ. Vegetatively growing or sporulating wild-type (AN120) and vps54Δ (Y6287) cells were harvested at indicated times, fixed with formaldehyde, and stained with DAPI to visualize nuclei and with rhodamine-phalloidine to visualize actin patches and cables. In wild-type cells, actin patches localized at the periphery of cells (0 hr) and spores (9 and 12 hr) and network-like actin filaments were detected in the cytoplasm of cells undergoing the meiotic divisions (3 and 6 hr). A similar pattern was observed in vps54Δ cells except some cells displayed actin clumps and there was an overall increase in cytoplasmic staining. Bar, 5 µm.

Figure 2 (.gif)

Supplementary Figure 3: Ether sensitivity of GARP mutants is not due to poor sporulation efficiency. spo14Δ cells, which do not form spores, were mixed with 40, 30, 20, 10, and 5% wild-type cells and spotted onto SPO plate. After 2-days incubation, ether sensitivity was examined as in Figure 5. A mixture of 5% wild-type spores was sufficient to survive ether treatment, indicating that the ether sensitivity of GARP mutants was not a consequence of poor sporulation (see in Table 3) but due to the formation of aberrant spores.

Figure 3 (.gif)