Abstract
We describe a regulatory mechanism that controls the activity of retromer, an evolutionarily conserved sorting device that orchestrates cargo export from the endosome. A spontaneously arising mutation that activates the yeast (Saccharomyces cerevisiae) CDC25 family phosphatase, Mih1, results in accelerated turnover of a subset of endocytosed plasma membrane proteins due to deficient sorting into a retromer-mediated recycling pathway. Mih1 directly modulates the phosphorylation state of the Vps26 retromer subunit; mutations engineered to mimic these states modulate the binding affinities of Vps26 for a retromer cargo, resulting in corresponding changes in cargo sorting at the endosome. The results suggest that a phosphorylation-based gating mechanism controls cargo selection by yeast retromer, and they establish a functional precedent for CDC25 protein phosphatases that lies outside of their canonical role in regulating cell cycle progression.
Highlights
Endocytosis mediates internalization of the plasma membrane via vesicles that deliver their content to the endosomal system, a network of related organelles that undergo maturation to generate a terminal endosome that fuses with the lysosome
Vps26 is structurally related to a- and b-arrestins (Collins et al, 2008; Shi et al, 2006), which function as sorting adapters for G protein-coupled receptors (GPCRs) in clathrin-mediated endocytosis, raising the
To identify factors that regulate post-Golgi trafficking of integral membrane proteins, we harnessed the activity of yeast chitin synthase 3 (Chs3), an integral membrane enzyme that is trafficked between Golgi and endosomal compartments and the plasma membrane
Summary
Endocytosis mediates internalization of the plasma membrane via vesicles that deliver their content to the endosomal system, a network of related organelles that undergo maturation to generate a terminal endosome that fuses with the lysosome. Given the central role that retromer plays in controlling plasma membrane composition and organelle biogenesis, it is logical to expect that retromer activities would be regulated in order to integrate its functions with cell physiology (Seaman, 2012), but examples of such regulation have yet to be identified. One mutant obtained displays a loss of retromer-dependent plasma membrane recycling of multiple integral plasma membrane proteins. This phenotype results from a gain-of-function mutation in the dual specificity protein phosphatase (Camps et al, 2000; Patterson et al, 2009), MIH1, encoding the S. cerevisiae homolog of CDC25 (Pal et al, 2008). We show that Vps directly recognizes a retromer sorting signal and that the phosphorylation state of Vps, controlled by Mih, modulates the affinity of retromer for the Chs recycling signal
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