Lessons from yeast for clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. As CME is highly conserved from yeast to humans, budding yeast provides an evolutionary template for this process and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system.

Figure 1

Yeast endocytic factors are comprised of many conserved modular domains. Shown are the major endocytic factors with domain structures. Each factor is listed in the order of its recruitment during endocytosis. A red * indicates the EH ligand NPF motifs and ‡ indicates the acidic motif needed for actin nucleation promoting activity.

Figure 2

Endocytic pathway in yeast. Early factors (Clathrin, Syp1, Ede1) are recruited during the immobile phase, which is followed by the ordered assembly of the mid/late coat (Sla2, Ap1801/, Ent1/2, Pan1, Sla1). Las17 is also recruited around this time. Shortly before the mobile phase Syp1 and Ede1 depart from the cortex and this is rapidly followed by the WASp/myosin/actin slow mobile invagination phase (Actin, Abp1, Arp2/3, Myo3/5, and Vrp1). Once the extended tubule forms, the vesicle scission apparatus (Rvs161/167 and Vps1) narrows the neck of the vesicle forming at the invagination tip to promote scission. Upon release, the nascent vesicle is immediately uncoated via synaptojanin and Prk1/Ark1 and then moves rapidly inward, shedding its actin shell via action of Cof1, Aip1, Srv2, and Crn1. Mid/late coat factors are reactivated via Scd5/PP1(Glc7) dephosphorylation and recruited back to the membrane for new rounds of CME.

Figure 3

Arp2/3 complex-mediated actin assembly is controlled temporally and spatially by many regulatory mechanisms during endocytosis. In this figure, NPFs are indicated in green, positive regulators of NPFs in blue and negative regulators in red. The colors of the negative regulators are changed to pink and then white to indicate their inhibitory activity is partial or relieved. F-actin is shown as a gray cloud surrounding the endocytic site. Initially actin assembly is blocked by Syp1, Sla1 and Sla2, which prevent early activation of Las17 and Pan1 (left). Also myosin 1 is inhibited by calmodulin in the cytosol. Next, the activator Bzz1 arrives and Vrp1 is recruited by Las17 (middle). This may allow early F-actin seeding and the beginning of membrane invagination, as inhibitors start to lose their influence (pink). Release of Syp1 from the cortex and recruitment of the myosins (by Vrp1) and Arp2/3 complex lead to robust actin assembly and invagination (right). Another negative regulator, Bbc1, arrives in the late phase, preventing excessive endocytic actin accumulation. It is not known when the Pan1 inhibitory function of Sla2 is relieved since they move together into the coat. Sla2 is shown here as being increasingly less influential as the endocytic site invaginates.

Figure 4

Translating actin assembly into membrane invagination is regulated by clathrin light chain. Sla2 is recruited to the cortex prior to actin assembly (I). As F-actin is assembled, Sla2 at the perimeter of the clathrin coat binds to the filaments through its C-terminal THATCH domain (II). This binding is used to tether the membrane to actin for invagination (III). As the clathrin coat continues to form at the tip, Sla2 is bound by clathrin LC causing a conformational shift (to closed) that releases Sla2’s hold on F-actin (IV). This may direct the force of actin assembly towards formation of a tubule, as well as promote vesicle scission at the neck.