Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis
Fig 5
The effect of the Vps1 KKK-AAA mutant on Vps1 function.
(A) Expression of wild type Vps1GFP and Vps1(KKK-AAA)-GFP mutant in cells. Western blot showing similar expression of wild type and mutant Vps1. GAPDH levels were used as a control for loading. (B) Processing of carboxypeptidase Y (CPY) requires trafficking to the vacuole. In the absence of Vps1 processing is defective and western blotting for the CPY protein demonstrates an accumulation of partially processed CPY protein (P). This accumulation is not seen in cells expressing wild type or the mutant Vps1 where only the mature (m) form is detected. (C) Localisation of GFP-Snc1-SUC2 fusion was analysed in a vps1 deletion strain and in the presence wild type Vps1 and Vps1 KKK-AAA mutant to investigate endocytic recycling of the SNARE protein. Scale bars 2 μM. (D) A Vps10-2x GFP Δvps1 strain was crossed with Δvps1 strain and strains expressing the wild type or Vps1 mutant to determine whether the mutant is able to rescue the retrograde trafficking phenotype. Scale bars 2 μM. (E) Peroxisomes labelled with GFP peroxisome reporter (GFP-SKL) were visualised in a Δvps1 Δdnm1 strain expressing plasmids with wild type or mutant versions of Vps1. Images are compressed Z-stacks for fission-proficient strains and in a single plane for fission-deficient strains. Scale bars 2 μM.