T-SNARE phosphorylation regulates endocytosis in yeast

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Molecular Biology of the Cell (Impact Factor: 4.47). 06/2002; 13(5):1594-607. DOI: 10.1091/mbc.01-11-0541
Source: PubMed


Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C(2)-ceramide, VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.

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    • "Molecules internalized by endocytic vesicles are transported to endosomal compartments , from which they can be recycled back to membranes or sent to degradative organelles such as lysosomes or vacuoles. Endocytosis has been well characterized in mammalian, yeast and plant cells by both biochemical and genetic dissection (Ayscough, 2005; Gurunathan et al., 2002; Kaksonen et al., 2003; Murphy et al., 2005; Smythe & Ayscough, 2006). Although there is substantial evidence that endocytosis occurs in (some) filamentous fungi, the issue is not without controversy (Read & Kalkman, 2003; Torralba & Heath, 2002). "
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    ABSTRACT: The entomopathogenic fungus Beauveria bassiana is under intensive study as a pest biological control agent. B. bassiana produces several distinct single-cell types that include aerial conidia, in vitro blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, B. bassiana displays a dimorphic transition, producing in vivo specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of in vitro and in vivo insect haemolymph-derived B. bassiana cells. In vitro blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected Heliothis virescens haemolymph (in vivo cells) actively internalized FM4-64. The in vivo blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.
    Microbiology 07/2009; 155(Pt 9):3110-20. DOI:10.1099/mic.0.029165-0 · 2.56 Impact Factor
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    • "Another way of increasing the peroxicretion efficiency is to use C2 ceramide. Activation of CAPP by adding C2-ceramide is known to result in increased availability of t-SNARE, Sso1p, which is important for SNARE-pin formation [26,27]. Indeed, addition of C2-ceramide slightly enhanced the peroxicretion efficiency (figure 12). "
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    ABSTRACT: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.
    BMC Biotechnology 06/2009; 9(1):48. DOI:10.1186/1472-6750-9-48 · 2.03 Impact Factor
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    • "II phosphorylates the synaptic Q a - SNARE syntaxin 1A in vitro ( Hirling and Scheller , 1996 ) . Phosphorylation often inversely correlates with fusion . Yeast cells expressing a Golgi Q a - SNARE Sed5 phosphomimetic mutant have fragmented Golgi compartments , whereas cells expressing a phosphorylation - resistant Sed5 mutant have ordered Golgi ( Gurunathan et al . , 2002 ; Weinberger et al . , 2005 ) . Rab effectors are also phosphorylated ( Lonart and Sudhof , 1998 ; Lonart et al . , 2003 ) . For example , phosphorylation of the Rab11 effector Rip11 controls its local - ization on apical recycling endosomes within epithelial cells ( Prekeris et al . , 2000 ) . Finally , the Ypt31 / 32 Rab GTPases regu "
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    ABSTRACT: Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.
    The Journal of Cell Biology 10/2008; 182(6):1141-51. DOI:10.1083/jcb.200801001 · 9.83 Impact Factor
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