Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases
Figure 2
PLP2 is required for the function of K5.
(A) PLP2 protein is not expressed in K5-PLP2GT cells. K5 and PLP2 were analysed by immunoblot from cell lysates of the indicated cell lines. (B) PLP2 is required for the K5-mediated downregulation of B7-2 in KBM7 cells. The K5-PLP2GT clone was transduced with a lentiviral vector expressing PLP2. Untransduced cells were removed by puromycin selection, and the surface expression of B7-2 in the indicated cell types was analysed by flow cytometry. Re-expression of PLP2 in K5-PLP2GT cells restores the K5-mediated downregulation of B7-2. Incomplete puromycin selection means that a small population of cells remain that are not transduced with PLP2, which therefore do not recover the K5-mediated downregulation of B7-2. (C) K5 requires PLP2 to downregulate a range of its targets. Cell surface expression of MHC-I, PECAM and ICAM-1 were analysed in the indicated cell types by flow cytometry. (D) HepG2 cells do not express detectable PLP2 protein. Immunoblot analysis of PLP2 from cell lysates of the indicated cell lines. (E) K5 cannot function in HepG2 cells owing to the absence of PLP2. Lentiviral expression of K5 alone in WT HepG2 cells does not affect cell surface MHC-I expression as measured by flow cytometry, but co-expression of K5 and PLP2 leads to downregulation of cell surface MHC-I.