Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER). They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES) on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae

Sar1 localizes at the rims of COPII-coated membranes in vivo

The Sar1 GTPase controls coat assembly on coat protein complex II (COPII)-coated vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi. The GTP-bound form of Sar1, activated by the ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by the COPII-vesicle-localized GTPase-activating protein (GAP) Sec23, in turn causes Sar1 to dissociate from the membrane. Thus, Sar1 is cycled between active and inactive states, and on and off vesicle membranes, but its precise spatiotemporal regulation remains unknown. Here, we examined Sar1 localization on COPII-coated membranes in living Saccharomyces cerevisiae cells. Two-dimensional (2D) observation demonstrated that Sar1 showed modest accumulation around the ER exit sites (ERES) in a manner that was dependent on Sec16 function. Detailed three-dimensional (3D) observation further demonstrated that Sar1 localized at the rims of the COPII-coated membranes, but was excluded from the rest of the COPII membranes. Additionally, a GTP-locked form of Sar1 induced abnormally enlarged COPII-coated structures and covered the entirety of these structures. These results suggested that the reversible membrane association of Sar1 GTPase leads to its localization being restricted to the rims of COPII-coated membranes in vivo

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell–derived VEGFR2+ mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)–BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A–induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras−/− mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2+ progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2+ progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6–9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras–Erk signaling to direct endothelial specification of VEGFR2+ vascular progenitor cells

Dynamic localization of a yeast development–specific PP1 complex during prospore membrane formation is dependent on multiple localization signals and complex formation

During the developmental process of sporulation in Saccharomyces cerevisiae, membrane structures called prospore membranes are formed de novo, expand, extend, acquire a round shape, and finally become plasma membranes of the spores. GIP1 encodes a regulatory/targeting subunit of protein phosphatase type 1 that is required for sporulation. Gip1 recruits the catalytic subunit Glc7 to septin structures that form along the prospore membrane; however, the molecular basis of its localization and function is not fully understood. Here we show that Gip1 changes its localization dynamically and is required for prospore membrane extension. Gip1 first associates with the spindle pole body as the prospore membrane forms, moves onto the prospore membrane and then to the septins as the membrane extends, distributes around the prospore membrane after closure, and finally translocates into the nucleus in the maturing spore. Deletion and mutation analyses reveal distinct sequences in Gip1 that are required for different localizations and for association with Glc7. Binding to Glc7 is also required for proper localization. Strikingly, localization to the prospore membrane, but not association with septins, is important for Gip1 function. Further, our genetic analysis suggests that a Gip1–Glc7 phosphatase complex regulates prospore membrane extension in parallel to the previously reported Vps13, Spo71, Spo73 pathway

Pop2 phosphorylation at S39 contributes to the glucose repression of stress response genes, HSP12 and HSP26

The S. cerevisiae Pop2 protein is an exonuclease in the Ccr4-Not complex that is a conserved regulator of gene expression. Pop2 regulates gene expression post-transcriptionally by shortening the poly(A) tail of mRNA. A previous study has shown that Pop2 is phosphorylated at threonine 97 (T97) by Yak1 protein kinase in response to glucose limitation. However, the physiological importance of Pop2 phosphorylation remains unknown. In this study, we found that Pop2 is phosphorylated at serine 39 (S39) under unstressed conditions. The dephosphorylation of S39 was occurred rapidly after glucose depletion, and the addition of glucose to the glucose-deprived culture recovered this phosphorylation, suggesting that Pop2 phosphorylation at S39 is regulated by glucose. This glucose-regulated phosphorylation of Pop2 at S39 is dependent on Pho85 kinase. We previously reported that Pop2 takes a part in the cell wall integrity pathway by regulating LRG1 mRNA; however, S39 phosphorylation of Pop2 is not involved in LRG1 expression. On the other hand, Pop2 phosphorylation at S39 is involved in the expression of HSP12 and HSP26, which encode a small heat shock protein. In the medium supplemented with glucose, Pop2 might be phosphorylated at S39 by Pho85 kinase, and this phosphorylation contributes to repress the expression of HSP12 and HSP26. Glucose starvation inactivated Pho85, which resulted in the derepression of HSP12 and HSP26, together with other glucose sensing mechanisms. Our results suggest that Pho85-dependent phosphorylation of Pop2 is a part of the glucose sensing system in yeast

Visualization of secretory cargo transport within the Golgi apparatus

To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone

A Screen for Spore Wall Permeability Mutants Identifies a Secreted Protease Required for Proper Spore Wall Assembly

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat

Cosmogenic beryllium 10 in ice cores and cosmic-ray stratigraphy

第2回極域科学シンポジウム　氷床コアセッション 11月16日（水） 国立極地研究所 2階大会議

Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation in Saccharomyces cerevisiae

The meiosis II outer plaque (MOP) acts a vesicle tethering complex that is a site for de novo membrane formation. Novel mutants in a MOP protein reveal that interaction of vesicles with the MOP surface is required to recruit a second tethering complex, the exocyst, to the vesicles, suggesting a mechanism by which the MOP promotes vesicle fusion