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: 4.55). 06/2002; 13(5):1594-607. DOI: 10.1091/mbc.01-11-0541
Source: PubMed

ABSTRACT 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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Available from: Adina Weinberger, Aug 17, 2015
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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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    • "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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    • "PKA is involved in the regulation of the Sso and Tlg t- SNAREs, which confer exo-and endocytic transport in yeast, respectively (Marash and Gerst, 2001; Gurunathan et al., 2002). In the case of the Sso t-SNAREs, phosphorylation inhibits t-t SNARE assembly and recruits a SNARE regulatory protein, Vsm1, to the autoinhibitory domain of Sso (Marash and Gerst, 2001; Marash and Gerst, 2003). "
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