Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis

Abstract

SummaryPhosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis. In this study we analyzed scd5-PP1D2, a mutation causing impaired PP1 binding. scd5-PP1D2 caused hyperphosphorylation of several Prk1 endocytic targets. Live-cell imaging of 15 endocytic components in scd5-PP1D2 revealed that most factors arriving before the invagination/actin phase of endocytosis had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homolog), whose lifetime was extended nearly fourfold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than twofold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1D2 were suppressed by SLA1 overexpression. This was dependent on the LxxQxTG repeats (SR) of Sla1, which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the dephosphorylated state. Without the SR, Sla1DSR was still recruited to the cell surface, but was less concentrated in cortical patches than Pan1. sla1DSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region the SH3 region of Sla1 causes constitutive negative regulation of Las17 (WASp). These results demonstrate that Scd5/PP1 is important for recycling Prk1 targets to initiate new rounds of endocytosis and provide new mechanistic information on the role of the Sla1 SR domain in regulating progression to the invagination/actin phase of endocytosis.

Structural Insights into Assembly and Regulation of the Plasma Membrane Phosphatidylinositol 4-Kinase Complex

“…Cortical patch to cytosol intensity ratio and patch density analysis were carried out as described (Chi et al, 2012). Briefly, the medial z plane from 3D image stacks of large budded cells was used to identify the brightest cortical patch in a cell, and the intensity of the brightest pixel was divided by fluorescence from the cell cytosol.…”

Phospho‐regulation of intrinsically disordered proteins for actin assembly and endocytosis

“…Deletion of the paralogous kinase genes ARK1 and PRK1 causes apparent deficiencies in disassembly of the endocytic coat [110,111], suggesting that phosphorylation possibly modulates the affinity among coat module proteins such as Sla1, Pan1 and Ent1/2. While the role of these kinases is less established for the WASP-myosin module, putative Prk1 consensus sites within Las17 have suggested a similar mechanism may also be involved [94,103,112]. Likewise, Scd5-targetted PP1 may increase affinity between module proteins, as has been suggested for coat module proteins [94,113,114].…”

Clathrin-mediated endocytosis in budding yeast at a glance

“…Scd5, a subunit that targets protein phosphatase 1 (PP1, Glc7 in yeast) (see poster), counteracts phosphorylation by Ark1 and Prk1 (Chang et al, 2002;Chi et al, 2012;Henry et al, 2003;Zeng and Cai, 1999;Zeng et al, 2007). In scd5 mutants that fail to interact with PP1/ Glc7, many endocytic adapter proteins in the coat module are hyperphosphorylated (Chi et al, 2012;Zeng et al, 2007).…”

“…In scd5 mutants that fail to interact with PP1/ Glc7, many endocytic adapter proteins in the coat module are hyperphosphorylated (Chi et al, 2012;Zeng et al, 2007). In addition, lack of Scd5 activity causes a delay in the transition from mid to late coat protein progression, which is likely to be due to inefficient recruitment of hyperphosphorylated Sla1 (Chi et al, 2012).…”