Figure 1.

Distribution of GFP-SifA in endocytic trafficking yeast mutants. (A) The distribution of GFP-SifA in fixed wild-type or endocytic trafficking deletion mutants was observed by direct fluorescence microscopy. Cells containing plasmids that expressed either the GFP-SifA fusion (pDV116) or GFP control were grown in media containing raffinose at 25°C. In all cases, except for vps1Δ cells, GFP-SifA localizes to distinct punctate structures (indicated by arrows). The diffuse cytoplasmic and nuclear fluorescence background was also observed in cells expressing only GFP. In vps1Δ mutants that lack the dynamin gene, GFP-SifA localizes to thin filamentous structures (arrowheads) anchored at the neck of large budded (G2) cells. Bar, 4 μm. (B) Introducing a plasmid expressing the wild-type dynamin VPS1 into vps1Δ cells was sufficient to redistribute GFP-SifA to punctate structures. Note that vps1Δ cells at other budding stages that harbor the control plasmid (vec) contain fewer (0–1 dot) and larger GFP-SifA punctate structures than those with the pVPS1 plasmid. Expression of GFP-SifA (pMO32) in vps1Δ cells was induced in galactose for 3 h, and fluorescent images were captured live. Bar, 4 μm.