Figure 7.

Reduction of PLAA Leads to Impaired Trafficking of K63-polyUb Substrates

(A and B) Affinity purification of P17 cerebellar lysates using the K63-specific UBD (EPS15) revealed a significant accumulation of K63-ubiquitylated proteins in Plaa^G23V/− mutants. Blot representative of n = 3 animals per genotype. Reduction of PLAA leads to increased binding of p62 to these increased K63-ubiqutitylated substrates (quantified in B).

(C–E) Plaa^−/− MEFs show significant increase in p62 foci (quantified in D), some of which colocalizes with mislocalized HRS (E) (see Figure S7G for zoomed out image). Scale bars represent 10 μm.

Error bars represent SEM; ns: not significant, ^∗p < 0.05, Student’s t test.

(F) Schematic representation of PLAA-dependent trafficking defects through the endolysosomal system. PLAA is required for sorting of Ub-modified membrane proteins into the lumen of MVB/late endosomes. Cargos become trapped on limiting membranes of abnormal early endosome intermediates in Plaa mutants where they are concentrated by p62 adaptor protein for alternate lysosomal degradation via autophagy. Alternately these proteins targeted for degradation may be re-routed via the recycling endosomes to the cell surface where they may be functionally compromised (red).

See also Figure S8.