Jiro Toshima

Endocytosis is the process by which cells internalize extracellular materials, cell surface proteins, and lipids via vesicles formed by membrane invagination. Endocytosed cargos are first delivered to sorting compartments and then recycled back to the plasma membrane or brought to late endosomal compartments en route to the lysosome/vacuole for degradation. The budding yeast Saccharomyces cerevisiae has been an important model organism for studying the molecular mechanisms involved in the multi-step process of the endocytic pathway, including clathrin-coated vesicle formation, vesicle internalization, cargo sorting, recycling, and degradation. In the yeast endocytic pathway, the mechanisms of formation and internalization of clathrin-coated vesicles are well conserved in relation to those in mammalian cells. Yeast also has mechanisms similar to those in mammalian cells for the regulation of events in the endo-lysosomal pathway, such as endosome maturation, multi-vesicular body formation, and retrograde transport. In contrast, the structure and properties of the endocytic sorting compartment in yeast differ significantly from those in mammalian cells. Recent studies have revealed that a sub-region within the trans-Golgi network (TGN) acts as a sorting compartment in yeast. In this chapter, we summarize recent findings regarding the molecular mechanisms of the endocytic pathway, particularly the involvement of the TGN.

After the endocytic and biosynthetic pathway converge, they partially share the route to the lysosome/vacuole. Similarly, the endocytic recycling and secretory pathways also partially share the route to the plasma membrane. The interaction of these transport pathways is mediated by endosomes and the trans-Golgi network (TGN), which act as sorting stations in endocytic and biosynthesis pathway, and endosomes has a bidirectional transport to and from the TGN. In mammalian cells endosomes can be largely classified as early/sorting, late, and recycling endosomes, based on their morphological features and localization of Rab family proteins, which are key factors in vesicular trafficking. However, these endosomes do not necessarily represent specific compartments that are comparable among different species. For instance, Rab5 localizes to early endosomes in mammalian cells but is widely localized to early-to-late endosomes in yeast, and to pre-vacuolar endosomes and the TGN in plant cells. The SNARE complexes are also key factors widely conserved among species and localized specifically to the endosomal membrane, but the localization of respective homologs is not necessarily consistent among species. These facts suggest that endosomes should be classified more inclusively across species. Here we reconsider the mammalian endosome system based on findings in budding yeast and other species and discuss the differences and similarities between them.

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN–endosome trafficking pathway.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Clathrin-mediated vesicle trafficking plays central roles in the post-Golgi transport pathways from the trans -Golgi network (TGN) to endosomes. In yeast, two clathrin adaptors – AP-1 complex and GGA proteins (GGAs) – are predicted to generate distinct transport vesicles at the TGN, and epsin-related Ent3p/Ent5p act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN, rather than from the plasma membrane, is crucial for Rab5-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGAs are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Rab5 activity remains ambiguous. Here we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Rab5 activation. We were able to show that AP-1 disruption in ent3 Δ/ 5 Δ mutant impairs Rab5-GEF Vps9p transport to the Rab5 compartment, and severely reduces Rab5 activity. Additionally, GGAs, Golgi-resident PI4 kinase Pik1p and Rab11 GTPases Ypt31p/32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Rab5 activation in the TGN-endosome trafficking pathway.

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood. In this study, to clarify the role of PS in the endocytic pathway, we analyzed the effect of PS depletion on endocytic internalization and post-endocytic transport. We demonstrated that in cell lacking the PS synthase Cho1p (cho1Δ cell), binding and internalization of mating pheromoneα-factor into the cell was severely impaired. Interestingly, the processes of endocytosis were mostly unaffected, but protein transport from the trans-Golgi network (TGN) to the PM was defective and localization of cell surface proteins was severely impaired in cho1Δ cells. We also showed that PS accumulated in intracellular compartments in cells lacking Rcy1p and Vps52p, both of which are implicated in endosome-to-PM transport via the TGN, and that the number of Snx4p-residing endosomes was increased in cho1Δ cells. These results suggest that PS plays a crucial role in the transport and localization of cell surface membrane proteins. Key Words: phosphatidylserine, endocytosis, recycling, vesicle transport

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells the trans -Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment, and function independently of the Sec7p-residing region within the TGN.

Endocytosis is a multistep process involving the sequential recruitment and action of numerous proteins. This process can be divided into two phases: an early phase, in which sites of endocytosis are formed, and a late phase in which clathrin-coated vesicles are formed and internalized into the cytosol, but how these phases link to each other remains unclear. In this study, we demonstrate that anchoring the yeast Eps15-like protein Pan1p to the peroxisome triggers most of the events occurring during the late phase at the peroxisome. At this ectopic location, Pan1p recruits most proteins that function in the late phases—including actin nucleation promoting factors—and then initiates actin polymerization. Pan1p also recruited Prk1 kinase and actin depolymerizing factors, thereby triggering disassembly immediately after actin assembly and inducing dissociation of endocytic proteins from the peroxisome. These observations suggest that Pan1p is a key regulator for initiating, processing, and completing the late phase of endocytosis.