Fig 5. Phosphorylation of specific VpsO C-terminal tail tyrosine residues is critical for VpsO function.

(A) Diagram of VpsO showing domain topology and the positions of the potential C-terminal tail phosphorylation sites. Signal peptide (SP), CytoPlasmic (CP), transmembrane (TM), Periplasmic (PP). CytoPlasmic Kinase Domain (CP-KD). (B) Colony corrugation phenotypes of the VpsO C-terminal tail mutants after 5 days of growth at 25°C. (C) Quantification of VPS in extracts of the C-terminal tail mutants compared to the rugose strain; n = 2 for biological replicates each with n = 3 for technical replicates. *****, p < 0.0005 by one-way Anova multiple comparisons test. (D) Western blot analysis for VpsO abundance and tyrosine phosphorylation; n≥3. (E) 50 μM VpsO-503 and the indicated three C-terminal tail mutants were incubated with [γ-³²P]-ATP for 30 minutes and autophosphorylation was analyzed by gel electrophoresis and phosphorimaging; n≥3.