Chi RJ, Torres OT, Segarra VA, Lansley T, Chang JS, Newpher TM, Lemmon SK

Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1/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-PP1Δ2, a mutation ...

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causing impaired PP1 binding. scd5-PP1Δ2 caused hyperphosphorylation of several Prk1 endocytic targets. Live cell imaging of 15 endocytic components in scd5-PP1Δ2 revealed most factors arriving before invagination/actin had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homologue), whose lifetime was extended nearly 4-fold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than 2-fold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1Δ2 were suppressed by SLA1 overexpression. This was dependent on Sla1's LxxQxTG repeats (SR), which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the de-phosphorylated state. Without the SR, Sla1ΔSR was still recruited to the cell surface, but was less concentrated in cortical patches as compared to Pan1. sla1ΔSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region Sla1's SH3 region 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.

Date: Jul. 23, 2012

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