Crosstalk between PI(4,5)P2and CK2 modulates actin polymerization during endocytic uptake.

A transient burst of actin polymerization assists endocytic budding. How actin polymerization is controlled in this context is not understood. Here, we show that crosstalk between PI(4,5)P2and the CK2 catalytic subunit Cka2 controls actin polymerization at endocytic sites. We find that phosphorylation of the myosin-I Myo5 by Cka2 downregulates Myo5-induced Arp2/3-dependent actin polymerization, whereas PI(4,5)P2cooperatively relieves Myo5 autoinhibition and inhibits the catalytic activity of Cka2. Cka2 and the PI(4,5)P2-5-phosphatases Sjl1 and Sjl2, the yeast synaptojanins, exhibit genetic interactions indicating functional redundancy. The ultrastructural analysis of plasma membrane invaginations in CK2 and synaptojanin mutants demonstrates that both cooperate to initiate constriction of the invagination neck, a process coupled to the remodeling of the endocytic actin network. Our data demonstrate a holoenzyme-independent function of CK2 in endocytic budding and establish a robust genetic, functional, and molecular link between PI(4,5)P2and CK2, two masters of intracellular signaling.

Identification and verification of Sro7p as an effector of the Sec4p Rab GTPase.

Effectors are operationally defined as proteins that recognize a specific GTPase preferentially in its GTP-bound conformation. Here we present the use of affinity chromatography to identify potential effectors of Sec4p, the Rab GTPase that controls the final stage of the yeast secretory pathway. We describe the preparation of the Rab protein affinity matrix and the yeast lysate used in the purification. We also describe the methods used to identify and verify one candidate, Sro7p, as a bona fide Sec4p effector. This includes tests of the specificity and efficiency of binding both in vitro and in vivo.

Rabs and their effectors: achieving specificity in membrane traffic.

Rab proteins constitute the largest branch of the Ras GTPase superfamily. Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. These different tasks are carried out by a diverse collection of effector molecules that bind to specific Rabs in their GTP-bound state. Recent advances have not only greatly extended the number of known Rab effectors, but have also begun to define the mechanisms underlying their distinct functions. By binding to the guanine nucleotide exchange proteins that activate the Rabs certain effectors act to establish positive feedback loops that help to define and maintain tightly localized domains of activated Rab proteins, which then serve to recruit other effector molecules. Additionally, Rab cascades and Rab conversions appear to confer directionality to membrane traffic and couple each stage of traffic with the next along the pathway.

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p.

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p.

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.