Coupled sterol synthesis and transport machineries at ER-endocytic contact sites.

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)-endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion.

ORP-Mediated ER Contact with Endocytic Sites Facilitates Actin Polymerization.

Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.

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.

Function and regulation of Saccharomyces cerevisiae myosins-I in endocytic budding.

Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-I Myo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites.

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.