Fig. 7: Rpc53 phosphorylation regulates V-ATPase-dependent autophagy.
a Schematic showing the phosphosites in Rpc53. The zoomed-in sequence shown with a proposed Kns1 motif (RXXS/TP) underlined and the phosphosite boxed in gray. Two overlapping GSK3 motifs (S/TXXXS/T) direct Mck1 phosphorylation of primed substrates (blue arrows) at phosphosites boxed in gray. Adapted from Lee et al., (2012). During nitrogen starvation or rapamycin treatment, Rpc53 is hyperphosphorylated and inactivated. As a result, ribosome biogenesis is repressed. b–d SEY6210 VMA2-AID*-9MYC cells (pRS307-CUP1p-GFP-ATG8) were grown to mid-log phase in YPD (0 h), centrifuged and resuspended in YPD + 0.1% DMSO, YPD + 300 μM 3-IAA, YPD + 200 nM rapamycin, or SD-N medium for the indicated times. Three biological replicates were repeated with similar results. b RPC53-TAP cell lysates were prepared and analyzed by western blot. Rapamycin treatment was the positive control that was known to induce complete Rpc53 phosphorylation. Rpc53-TAP protein was detected with anti-PAP antibody. The upper band represents phosphorylated Rpc53 (p-Rpc53-TAP) with lower mobility on an SDS-PAGE gel. c SEY6210 RPC53-TAP and RPC53T228A-TAP cell lysates were processed and analyzed by western blot. Gcn4 protein was detected with an anti-Gcn4 antibody. *, non-specific band. d RPC53-TAP and RPC53T228A-TAP cells transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-3xHA plasmid were harvested, processed, and analyzed by western blot. e Graphical model of V-ATPase-dependent autophagy. 1 A, FL-associated mutations of the V-ATPase subunits cause vacuolar deacidification. 1 B, unlike nitrogen starvation-induced autophagy, the TORC1 complex remains active in this context. 2 A, The Gcn2-Gcn4 pathway is activated by the vacuolar deacidification signal. 2B, Rpc53 is partially phosphorylated by Kns1 and Mck1 and thus inactivated. This induces a downregulation of ribosome biogenesis. 2 C, the downregulation of ribosome biogenesis releases the inhibitory effect on Gcn4 translational activation, thus further stimulating the Gcn2-Gcn4 pathway. 3 A, Trp prototrophy partially inhibits Rpc53 phosphorylation, and therefore maintains ribosome biogenesis in an active state. 3B, in addition, the Trp biosynthetic pathway fuels the intracellular NAD+ pool, which inhibits V-ATPase-dependent autophagy without affecting Gcn4 induction.
