The Yeast Arf GTPase-activating Protein Age1 Is Regulated by Phospholipase D for Post-Golgi Vesicular Transport
ABSTRACT Vesicular transport shuttles cargo among intracellular compartments. Several stages of vesicular transport are mediated by
the small GTPase Arf, which is controlled in a cycle of GTP binding and hydrolysis by Arf guanine-nucleotide exchange factors
and Arf GTPase-activating proteins (ArfGAPs), respectively. In budding yeast the Age2 + Gcs1 ArfGAP pair facilitates post-Golgi
transport. We have found the AGE1 gene, encoding another ArfGAP, can in high gene-copy number alleviate the temperature sensitivity of cells carrying mutations
affecting the Age2 + Gcs1 ArfGAP pair. Moreover, increased AGE1 gene dosage compensates for the complete absence of the otherwise essential Age2 + Gcs1 ArfGAP pair. Increased dosage of
SFH2, encoding a phosphatidylinositol transfer protein, also allows cell growth in the absence of the Age2 + Gcs1 pair, but good
growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase
D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by
lipid composition and can provide ArfGAP function for post-Golgi transport.
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- "The Sorting nexin family (Table 3) regulates endosomal sorting of internalized proteins through modulating endosomal-to-lysosomal sorting (Carlton et al., 2004). Both elevated Arf GTPase (Tables 1 and 3) or attenuated Arf GTPase activating protein family (Table 4) expression may help maintain Arf GTPase activity which is essential for the recruitment of coat protein complex I and clathrin coat complexes needed for the generation of transport vesicles (Benjamin et al., 2011). "
ABSTRACT: Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL's role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies.Frontiers in Immunology 02/2012; 3(15). DOI:10.3389/fimmu.2012.00015
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- "Arl1-His6 and Arf1-His6 were each coexpressed with N-myristoyltransferase in BL21(DE3) bacterial cells and purified following the procedure to isolate Arf1-His6 as described (Benjamin et al., 2011). Myristoylated GTPase was loaded with radioactive GTP, and GAP activity was assessed as described (Huber et al., 2001; Poon et al., 2001). "
ABSTRACT: Small monomeric G proteins regulated in part by GTPase-activating proteins (GAPs) are molecular switches for several aspects of vesicular transport. The yeast Gcs1 protein is a dual-specificity GAP for ADP-ribosylation factor (Arf) and Arf-like (Arl)1 G proteins, and also has GAP-independent activities. The absence of Gcs1 imposes cold sensitivity for growth and endosomal transport; here we present evidence that dysregulated Arl1 may cause these impairments. We show that gene deletions affecting the Arl1 or Ypt6 vesicle-tethering pathways prevent Arl1 activation and membrane localization, and restore growth and trafficking in the absence of Gcs1. A mutant version of Gcs1 deficient for both ArfGAP and Arl1GAP activity in vitro still allows growth and endosomal transport, suggesting that the function of Gcs1 that is required for these processes is independent of GAP activity. We propose that, in the absence of this GAP-independent regulation by Gcs1, the resulting dysregulated Arl1 prevents growth and impairs endosomal transport at low temperatures. In cells with dysregulated Arl1, an increased abundance of the Arl1 effector Imh1 restores growth and trafficking, and does so through Arl1 binding. Protein sequestration at the trans-Golgi membrane by dysregulated, active Arl1 may therefore be the mechanism of inhibition.Molecular biology of the cell 05/2011; 22(13):2337-47. DOI:10.1091/mbc.E10-09-0765 · 5.98 Impact Factor
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ABSTRACT: The protein cargo transported by specific types of vesicles largely defines the different secretory trafficking pathways operating within cells. However, mole per mole the most abundant cargo contained within transport vesicles is not protein, but lipid. Taking a "lipid-centric" point-of-view, we examine the importance of lipid signaling, membrane lipid organization and lipid metabolism for vesicle transport during exocytosis in budding yeast. In fact, the essential requirement for some exocytosis regulatory proteins can be bypassed by making simple manipulations of the lipids involved. During polarized exocytosis the sequential steps required to generate post-Golgi vesicles and target them to the plasma membrane (PM) involves the interplay of several types of lipids that are coordinately linked through PI4P metabolism and signaling. In turn, PI4P levels are regulated by PI4P kinases, the Sac1p PI4P phosphatase and the yeast Osh proteins, which are homologs of mammalian oxysterol-binding protein (OSBP). Together these regulators integrate the transitional steps required for vesicle maturation directly through changes in lipid composition and organization.07/2012; 2(3):151-160. DOI:10.4161/cl.20490