Kyoungtae Kim

The yeast dynamin-like protein Vps1:vps1 mutations perturb the internalization and the motility of endocytic vesicles and endosomes via disorganization of the actin cytoskeleton

Mammalian dynamin is responsible for scission of endocytic vesicles from the plasma membrane. A previous study showed that Vps1, a yeast dynamin-like protein, plays an important role in pheromone receptor internalization (Yu and Cai, 2004; J. Cell Sci. 117, 3839-3853). However, the details of how Vps1 acts in various phases of endocytosis including early internalization of the endocytic vesicle are poorly understood. To investigate the potential roles of Vps1 in both endocytic vesicle formation/maturation on the plasma membrane and endocytic vesicle internalization, time-lapse fluorescent images of GFP-tagged endocytic markers in live cells were analyzed using a particle tracking software. The loss of Vps1 leads to a robust increase in the lifespan of newly forming cortical endocytic vesicles carrying Las17-GFP, Ede1-GFP, Sla1-GFP, and Abp1-GFP, indicating that Vps1 is required for the proper assembly and maturation of endocytic vesicles. Particle track analysis revealed that Abp1-GFP vesicles in vps1 null cells moved a relatively short distance away from the cell membrane due to their non-directional movement. Furthermore, we found that the GTPase and the GED domains of Vps1 are required for the proper endocytic function of Vps1. Our tracking analysis data also revealed that the post-internalized vesicle motility en route to the vacuole was decreased significantly, perhaps due to severe disruption of the actin cables in Vps1 mutant cells.

Pil1, an eisosome organizer, plays an important role in the recruitment of synaptojanins and amphiphysins to facilitate receptor-mediated endocytosis in yeast

The eisosome protein Pil1 is known to be implicated in the endocytosis of Ste3, but the precise biological function of it during endocytosis is poorly understood. Here, we present data to reveal Pil1s role in receptor-mediated endocytosis. Using live cell imaging, we show that endocytic patches carrying Abp1 and Las17 persisted much longer in PIL1-deficient cells. The loss of Pil1 also greatly affected both the scission efficiency and the frequency of formation of endocytic sites carrying Rvs161- and Rvs167-GFP. Furthermore, the mistargeting of the synaptojanins, Sjl1 and Sjl2, to the cytoplasm in pil1Δ cells suggests that Pil1 is required for the proper recruitment of the synaptojanins to endocytic sites. A severe motility defect of Abp1-GFP during its internalization in a codeletant of PIL1 and SJL2 indicates a functional interplay between them in endocytosis. Together, these results establish that Pil1 is involved in the recruitment of endocytic proteins to optimize endocytosis.

Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae

Eisosomes are large immobile assemblies at the cortex of a cell under the membrane compartment of Can1 (MCC) in yeast. Slm1 has recently been identified as an MCC component that acts downstream of Mss4 in a pathway that regulates actin cytoskeleton organization in response to stress. In this study, we showed that inactivation of Slm proteins disrupts proper localization of the primary eisosome marker Pil1, providing evidence that Slm proteins play a role in eisosome organization. Furthermore, we found that slmts mutant cells exhibit actin defects in both the ability to polarize cortical F-actin and the formation of cytoplasmic actin cables even at the permissive temperature (30°C). We further demonstrated that the actin defect accounts for the slow traffic of FM4-64-labelled endosome in the cytoplasm, supporting the notion that intact actin is essential for endosome trafficking. However, our real-time microscopic analysis of Abp1-RFP revealed that the actin defect in slmts cells was not accompanied by a noticeable defect in actin patch internalization during receptor-mediated endocytosis. In addition, we found that slmts cells displayed impaired membrane recycling and that recycling occurred in an actin-independent manner. Our data provide evidence for the requirement of Slm proteins in eisosome organization and endosome trafficking and recycling.

Retromer: Structure, function, and roles in mammalian disease

Retrograde transport from the endosome to the Golgi is mediated by a 5 protein complex known as the retromer. These five proteins (Vps5, Vps17, Vps26, Vps29, and Vps35 in yeast and SNX1/2, SNX5/6, Vps26, Vps29, and Vps35 in mammalian cells) act as a coat for vesicles budding off of the endosome, as well as perform cargo sorting at the endosome. The retromer is well conserved between yeast and mammalian systems, though variations exist within the mammalian retromer. Functionally, the retromer has been linked to prominent neurodegenerative diseases such as Alzheimers and Parkinsons in human models as well as diabetes mellitus. However, the retromer also plays a role in the virulence of several microbial pathogens. Despite the current understanding of the retromer complex, there are still many questions to be answered in regards to its overall role in cell homeostasis.

From membranes to organelles: emerging roles for dynamin-like proteins in diverse cellular processes.

Dynamin is a GTPase mechanoenzyme most noted for its role in vesicle scission during endocytosis, and belongs to the dynamin family proteins. The dynamin family consists of classical dynamins and dynamin-like proteins (DLPs). Due to structural and functional similarities DLPs are thought to carry out membrane tubulation and scission in a similar manner to dynamin. Here, we discuss the newly emerging roles for DLPs, which include vacuole fission and fusion, peroxisome maintenance, endocytosis and intracellular trafficking. Specific focus is given to the role of DLPs in the budding yeast Saccharomyces cerevisiae because the diverse function of DLPs has been well characterized in this organism. Recent insights into DLPs may provide a better understanding of mammalian dynamin and its associated diseases.

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.

Vps1, a recycling factor for the traffic from early endosome to the late Golgi.

Recycling of cellular membranes and their constituents plays a role for cell survival and growth. In the budding yeast, there are recycling traffics from early and late endosomal compartments to the late Golgi. Here, we examined a possible role for Vps1, a large GTPase, in the recycling traffic of GFP-Snc1 from early endosomes to the late Golgi. In the absence of Vps1 we observed an aberrant accumulation of GFP-Snc1 puncta in the cytoplasm that we identified as early endosomes. The N-terminal GTPase and the C-terminal GED domains of Vps1 are essential for Vps1s function in Snc1 recycling. Our finding of genetic interactions of VPS1 with genes involved in early endosome-to-Golgi traffic further suggests Vps1 functions as a recycling factor in the membrane traffic. Finally, we provide evidence that the severe accumulation of GFP-Snc1 cytoplasmic puncta in vps1Δ cells is attributed to a mild defect in the retention of the GARP component Vps51 at the late Golgi, as well as a severe disruption of actin cables.

Insights into eisosome assembly and organization

Eisosomes, large protein complexes that are predominantly composed of BAR-domain-containing proteins Pil1 and its homologs, are situated under the plasma membrane of ascomycetes. A successful targeting of Pil1 onto the future site of eisosome accompanies maturation of eisosome. During or after recruitment, Pil1 undergoes self-assembly into filaments that can serve as scaffolds to induce membrane furrows or invaginations. Although a consequence of the invagination is likely to redistribute particular proteins and lipids to a different location, the precise physiological role of membrane invagination and eisosome assembly awaits further investigation. The present review summarizes recent research findings within the field regarding the detailed structural and functional significance of Pil1 on eisosome organization.

Molecular dynamics at the endocytic portal and regulations of endocytic and recycling traffics.

Endocytic and recycling pathways involve the transportation of soluble and transmembrane cargos to destinations within the cell or back to the plasma membrane for reuse. Common mechanistic themes for the traffic pathways in eukaryotic cells from yeast to mammalian cells are well-conserved, manifested by the molecular choreography of cargo segregation, membrane budding and coating, pinching off of the invaginated vesicle, cytoskeleton-mediated vesicle motility and fusion with target compartments. Here, we discuss recent insights into the spatiotemporal dynamics of endocytic machinery at the plasma membrane and the molecular details of bifurcating traffics at the endosome either to the lysosome or to the trans-Golgi network (TGN).

Insight into Tor2, a budding yeast microdomain protein.

The plasma membrane of eukaryotic organisms is compartmentalized into microdomains. The budding yeast Saccharomyces cerevisiae presents three laterally distinct microdomains: membrane compartment containing Can1 (MCC), membrane compartment containing Pma1 (MCP), and membrane compartment containing Tor2 (MCT). Tor2 and its corresponding protein complex, target of rapamycin complex 2 (TORC2), has been of particular interest in recent years. Tor2, the main organizer of TORC2, is a highly conserved kinase that has proved to be an important regulator of multiple cellular functions including cell growth, actin polymerization, endocytosis, and sphingolipid synthesis. Despite significant advancements, the full understanding of the Tor2 signaling networks is incomplete. Here we review the most compelling evidences for the function and physiological significance of Tor2, as well as discuss possible implications and explanations for observed phenomena.