Internalization in yeast: general requirements

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Receptor-mediated endocytosis in S. cerevisiae has been studied for two G protein serpentine receptors, the a and a-factor receptors, both of which display constitutive and ligand-induced endocytosis [27]. The endocytosis of cell-surface transporters has also been thoroughly studied in this organism. These transporters display both constitutive and accelerated endocytosis, specifically regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions [50].

Although yeast cells have no caveolin, and therefore no caveolae-dependent endocytosis, there is still some debate concerning whether clathrin-dependent endocytosis occurs in this organism (reviewed in [51]). Clathrin-coated vesicles have been purified from S. cerevisiae. However, attempts to visualize clathrin-coated pits and clathrin-coated vesicles at the yeast plasma membrane have not been successful [51]. Deletions of the genes encoding the clathrin light or heavy chains, or heat-sensitive mutations in the first of these genes, result in only 30-50% inhibition of internalization of the a-factor and a-factor receptors [52]. A clear difference between yeast and animal cells in the internalization step of endocytosis is the lack of involvement of dynamin, endophilin and AP-type adaptors in yeast. Yeast cells contain genes encoding three AP complexes, known as the AP1, AP2-like and AP3 adaptors. The AP2-like adaptor complex is not only dispensable for endocytosis, but may even fail to associate with clathrin in yeast cells [53, 54].

The other requirements for endocytosis have been investigated by studying several available mutants and screening to identify endocytic mutants. A number of proteins specifically required for the internalization step of endocytosis have been identified in this way (Table I). The correct organization of the actin cytoskeleton is a key requirement for endocytosis in yeast (reviewed in [27]). Many of the first end (for endocytosis) mutants identified were found to have mutations in the actin gene itself (END7) [55], in genes encoding actin-binding proteins (END6/RVS161, END5/VRP1) or in genes encoding proteins required for the correct organization of the actin cytoskeleton (e.g. END3, END4/SLA2, ARP2, END9/ARC35). The precise role of actin in yeast endocytosis is still unclear but, several years after the first study with yeast mutants [56], it was demonstrated using latrunculin A, a drug that sequesters actin monomers that correct actin polymerization is also required for endocytosis in mammals [57]. It has been suggested that, in yeast, actin polymerization may provide, in conjunction with the myosin isoforms Myo3p and Myo5p, the force required for the fission of vesicles from the plasma membrane (reviewed in [27]). This suggestion was based, in part, on the lack of involvement of dynamin-like proteins in yeast endocytosis. The two homologs of amphiphysin, Rvs161p and Rvs167p (Fig. 1), were identified among the proteins required for endocytosis in yeast. Cells defective in these proteins have impaired actin cytoskeleton organization. As mammalian amphiphysin interacts with dynamin, Rvs167p interacts with actin [58]. Amphiphysin/Rvs proteins are among the actors in endocytosis in both yeast and mammals, with apparent adaptation in their respective roles. Anyhow, the actin cytoskeleton now clearly appears involved in the internalization process in mammalian cells. Several clathrin-coated pits associated proteins, including dynamin, seem to play a role in regulating actin polymerization, and actin accumulation was observed at clathrin spots while they were disappearing. Actin was thus proposed to provide a force during vesicle formation, possibly to push the nascent vesicle away from the plasma membrane [59, 60].

Genetic screening, in addition to identifying mutations in genes related to actin function, also revealed the crucial role of lipids in endocytosis [27], and led to the identification of several clathrin-binding proteins essential for endocytosis. These proteins include Ent1p and Ent2p -the yeast homologs of epsin- and Pan1p, a protein carrying two EH domains. Yeast cells carry several EH domain-containing proteins, including Ede1p, a protein that like Eps15 carries 3 N-terminal EH domains, but has no DPF motifs (Fig. 1). Ede1p has been shown to be required for efficient endocytosis [61]. Extensive genetic studies and two-hybrid analysis led to the proposal that a complex network of interacting proteins linked to the actin cytoskeleton, including End3p (one EH doman), Pan1p and its Ent1/2 partners [62], is involved in endocytosis in yeast [63]. Ede1p may also be part of this complex, given genetic interactions between EDE1, PAN1, and END3 [61]. This suggestion was based on the analogy between this complex of EH domain-containing proteins with associated partners and the network of EH domain proteins and their partners involved in endocytosis in mammalian cells, the function of which appears to be more clearly defined [36]. The discovery that some of the corresponding yeast (Ede1p, Ent1/Ent2p), and mammalian proteins (Eps15, Epsins) contain ubiquitin-binding domains (Fig. 1), and improvements in our understanding of the role of ubiquitylation events in yeast and mammalian endocytosis have led to the formulation of new hypotheses concerning the possible function of some of the proteins in these complexes as adaptors, as detailed below.