Fig 1.
Highly simplified model of Sce endocytic vesicle formation.
Cargo selection recruits early coat proteins (red, Ede1p) and clathrin to endocytic sites. Additional coat proteins are recruited (dark blue, Sla1/2p), then actin and actin polymerization machinery (Las17p, Abp1p). Additional actin polymerization in catalyzed by Sac6p, and vesicles are scissioned via Rvs161p activity. Residence times listed for each protein are based on [41].
Fig 2.
Wbm0076 disrupts late endocytic vesicle kinetics, but not site selection.
Yeast strains harboring indicated GFP-tagged endocytic vesicle components, Abp1p-mCherry, and either pYES2/NT A (vector) or wBm0076 expression vector were induced for 5 h with 1 μM β-estradiol. Cells were harvested, washed, and (A) visualized via epifluorescent microscopy. Right panels are corresponding strains without β-estradiol induction. White arrows point to coupled (vector) or uncoupled (wBm0076) phenotype. (B) The percentage of cells showing colocalized patches in Sla1GFP + Abp1RFP strains with vector or wBm0076 expression in (A) were calculated (n ≥ 100 cells, in triplicate). (C) Yeast strains harboring Sla1-GFP and Abp1-mCherry and either a vector control or wBm0076 were induced for 5h in 1μM β-estradiol and visualized via two-color TIRF microscopy over 352 s (3 frames/s). Arrows point to actin patch analyzed by corresponding kymograph. (D) Kymograms describing protein kinetics of all patches visualized in a single cell. Bar = 5 μ.
Fig 3.
Wbm0076 domain mutations modify toxic activity.
Yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters (Methods), expressing Abp140GFP and harboring a pYES2/NT A control plasmid or a pYES2/NT A plasmid containing wBm0076 or mutant derivative were grown overnight in CSM medium lacking uracil. (A) Cultures were diluted to an OD600 = 1.0 in sterile 0.9% NaCl, then spotted in 10-fold dilutions on plates containing or lacking 1 μM β-estradiol. (B) Cells were subcultured to fresh CSM-ura containing or lacking 1 μM β-estradiol. After 5 h outgrowth at 30°C, cells were harvested and visualized. Bar = 5 μ. Number of colocalized actin patches (white arrows) and standard deviation from norm per cell is noted and determined from three independent experiments: n ≥ 100 cells per experiment. P > 0.05 (n.s.).
Fig 4.
Wbm0076 activity requires membrane association.
Yeast strains modified with GEV for β-estradiol-dependent induction of GAL promoters expressing Abp1RFP and harboring a pYES2/NT A control plasmid or a pYES2/NT A plasmid containing the indicated construct were grown overnight in CSM medium lacking uracil. (A) Cultures were diluted to an OD600 = 1.0 in sterile 0.9% NaCl, then spotted in 10-fold dilutions on plates containing or lacking 1 μM β-estradiol. (B) Cells were subcultured to fresh CSM-ura containing or lacking 1 μM β-estradiol. After 5 h outgrowth at 30°C, cells were harvested and visualized. Number of cells with colocalized actin patches (white arrows) and the standard deviation from the norm is noted and determined from three independent experiments: n ≥ 100 cells per experiment, bar = 5 μ.
Fig 5.
Wbm0076 coprecipitates with GST-Abp1p.
Protein extracts from indicated strains were generated and incubated with glutathione beads as in Methods. Equal fractions of input and elution for each condition were separated via SDS-PAGE and immunoblotted with the indicated antibody (Anti-Xpress: Wbm0076; anti-GST, GST-Abp1). Due to the similarity in the sizes of GST-Abp1 and Wbm0076, two identical gels and membranes were probed with different primary antibodies.
Fig 6.
Wbm0076 is conserved among Wolbachia endosymbionts.
Protein sequences from the Wolbachia genus homologous to Wbm0076 (only 22 of 31 shown here) were identified via the Blastp suite [91] and are indicated with abbreviations of the species of origin. Sequence alignment of conserved domains are highlighted with a black box. TM = transmembrane; V = verprolin/WH2 domain; C = central domain; A = acidic domain; P/PP = polyproline motif. Species abbreviations and NCBI accession numbers for all 31 identified homologues are available in S5 Fig.