A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor
Fig 5
Pair-wise comparisons of the proximity-omes of wild type Vpu relative to the ER-retained Vpu-A18H and the plasma-membrane-enriched Vpu-S52,56N mutants.
HeLa P4.R5 cells were transfected to express Vpu constructs bearing C-terminal APEX2 tags, in duplicate. Following proximity biotinylation reactions, the biotinylated proteins were isolated and subject to quantitative mass spectrometry. Volcano plots of protein enrichment in the presence of Vpu mutants A18H or S52,56N relative to the wild-type Vpu are shown, n = 2 experiments. Significantly enriched proteins highlighted in red and blue are derived from the Student’s t-test (p < 0.05). (A) The A18H mutant was enriched in proteins derived from the biosynthetic pathway, including the ER (labeled), while WT Vpu was enriched for proteins associated with plasma and endosomal membranes (labeled). (B) The S52,56N mutant was enriched in proteins derived from the plasma membrane (labeled), while WT Vpu was again enriched in endosomal sorting proteins (labeled). Proteins enriched by the S52,56N mutation included the known targets CD4, CD81, and HLA-C, and possible targets EGFR and CD55. For both (A) and (B), the x-axis shows log2 fold change of proteins enriched by mutant/WT Vpu and the y-axis -log10 of p-value derived from Student’s t-test. The 10 most highly enriched gene ontology (cellular component) terms are shown on the right of each volcano plot, corresponding to significantly enriched proteins proximal to the mutants; p-value derived from Bonferroni test.