Clathrin-mediated endocytosis in budding yeast

Abstract

Clathrin-mediated endocytosis in the budding yeast Saccharomyces cerevisiae involves the ordered recruitment, activity and disassembly of nearly 60 proteins at distinct sites on the plasma membrane. Two-color live-cell fluorescence microscopy has proven to be invaluable for in vivo analysis of endocytic proteins: identifying new components, determining the order of protein arrival and dissociation, and revealing even very subtle mutant phenotypes. Yeast genetics and functional genomics facilitate identification of complex interaction networks between endocytic proteins and their regulators. Quantitative datasets produced by these various analyses have made theoretical modeling possible. Here, we discuss recent findings on budding yeast endocytosis that have advanced our knowledge of how -60 endocytic proteins are recruited, perform their functions, are regulated by lipid and protein modifications, and are disassembled, all with remarkable regularity.

Figure 1. Actin structures in budding yeast

In budding yeast actin forms three structures: the cytokinetic ring (not shown) responsible for separating the mother and daughter cell cytoplasm; actin cables, which are parallel bundles of short actin filaments running the length of the cell, nucleated by formins and serving as tracks for myosin-based motility (green); and cortical actin patches, composed of branched filaments nucleated by the Arp2/3 complex, and which are the sites of endocytosis (red). Cables and patches are visualized by imaging Abp140-3xGFP and Abp1-RFP, respectively.

Figure 2. Timeline for endocytic vesicle formation and modular organization of proteins

Endocytic proteins are dynamically recruited in a highly predictable order. The “early” and “early coat” proteins are present at the cell surface for a variable amount of time. After a transition point, possibly defined by cargo recruitment, the lifetimes of endocytic proteins are quite regular. Actin polymerization and BAR domain proteins bend the membrane into an extended tubule, which is pinched off by the combined actions of BAR domain proteins, actin polymerization and possibly by a lipid phase separation. The newly formed vesicle is uncoated by the combined actions of the Ark1p/Prk1p kinases, the Sjl2p lipid-phosphatase and the actions of Arf3p/Gts1p/Lsb5p.

Figure 3. Endocytic mutants affecting actin polymerization

A. In sla1Δ bbc1Δ mutants, Las17p inhibition is greatly reduced, resulting in excessive actin polymerization. The connection between actin and the endocytic coat is intact, so deep invaginations are formed. B. In sla2Δ mutants the connection between actin and the endocytic coat is missing, resulting in long, treadmilling actin tails that continuously assemble proximal to the plasma membrane, and flat membranes. Coat proteins and NPFs remain at the plasma membrane while all the actin-associated proteins normally associated with endocytosis localize to the comet tails.

Figure 4. Scission and uncoating of endocytic vesicles

Scission is accomplished by constriction of the bud neck, driven by BAR protein-driven membrane deformation, actin-generated force and the proposed Sjl2p-imposed line tension created by lipid phase separation. After scission, the Pan1 complex proteins Sla1p, Pan1p and End3p are phosphorylated by Ark1p/Prk1p resulting in their dissociation. Ark1p/Prk1p are also responsible for turning off actin polymerization, allowing Cof1p/Aip1p/Crn1p to disassemble the actin filaments. Sjl2p is responsible for dephosphorylating PIP2, reducing the affinity of Sla2p and Ent1/2p for the vesicles. Arf3p, along with Gts1p and Lsb5p, are also involved in the dissociation of the Pan1 complex.

Figure 5. Positive feedback loops involved in scission

During scission, two positive feedback loops are proposed to drive the increase in membrane curvature that leads to pinching off of the vesicle. Firstly, BAR domain proteins bind to areas of high membrane curvature generated by actin polymerization forces, and through their binding enhance the membrane curvature, recruiting more BAR domain proteins. Secondly, Sjl2p, the PIP2 phosphatase, is more active on curved membranes [83]. The activity of Sjl2p on the curved neck decreases levels of PIP2 on the unprotected bud while the PIP2 of the neck is shielded by the BAR domain proteins. The lipid phase separation produces a line tension, which further increases membrane curvature, enhancing the activity of Sjl2p.