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  • Here we show in contrast to previous work that several

    2022-06-15

    Here we show, in contrast to previous work, that several phenotypes associated with Orb6 inactivation, including increased cell width, are independent of Gef1. We further find that Orb6 positively regulates exocytosis, also independently of Gef1. To identify novel targets of Orb6, we performed quantitative global phosphoproteomics analyses of desipramine sale with decreased Orb6 kinase activity and generated a high-quality dataset of Orb6 targets in vivo. Targets include proteins involved in kinase signaling, membrane trafficking, and the exocyst complex, a conserved octameric complex involved in late stages of exocytosis (Kee et al., 1997, TerBush and Novick, 1995). The exocyst, which contains proteins that bind to secretory vesicles, as well as proteins that bind to the plasma membrane, mediates secretory vesicle docking to the plasma membrane before SNARE-mediated vesicle fusion (Heider and Munson, 2012, Wu and Guo, 2015). We show that Orb6 phosphorylates exocyst proteins in vitro and that exocyst phosphorylation in vivo mediates a subset of Orb6-dependent phenotypes. Overall, our results suggest that Orb6 regulates cell polarity via multiple targets involved in membrane trafficking and exocytosis.
    Results
    Discussion Here we have shown that the conserved NDR/LATS kinase Orb6 has a major role in regulating fission yeast exocytosis. Round and/or wide cell-polarity defects in orb6 mutants were previously attributed to failure to spatially restrict activity of Cdc42 GEF Gef1 (Das et al., 2009). However, we find that these and several other orb6 phenotypes persist in gef1Δ cells. Upon inhibition of Orb6 kinase activity in vivo, impaired exocytosis is a key Gef1-independent phenotype, and from an unbiased quantitative global phosphoproteomics analysis, we identified several novel Orb6 targets related to membrane trafficking, including proteins implicated in signaling, lipid binding, and exocytosis. Through more detailed analysis of Orb6 phosphorylation of the exocyst complex, we have shown that Sec3 serine-201 phosphorylation is important for exocyst function.
    STAR★Methods
    Acknowledgments We thank M. Balasubramanian, D. McCollum, T. Nakamura, P. Nurse, F. Verde, P. Perez, J.C. Ribas, and H. Snaith for strains; I. Hagan and A. Patel for advice and protocols on phosphoproteomics; H. Thakur and S.L. Leong for advice on protein purification; and members of our laboratories for discussion. This work was supported by the BBRSC (BB/K021699/1 to K.E.S. and A.B.G.) and by the Wellcome Trust (094517 and 210659 to K.E.S., 108504 and 091020 to J.R., and a multi-user equipment grant 101527 for the Edinburgh Protein Production Facility). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149).
    Overview of the SV cycle and the AZ Communication between nerve cells largely occurs at synapses, specialized contact sites between neurons where electrical signals trigger the release of neurotransmitters, which in turn activate postsynaptic receptors. Efficient synaptic signaling is crucial for brain function, especially for memory storage and the processing of sensory information. Neurotransmitter release is mediated by the Ca2+-triggered exocytic fusion of synaptic vesicles (SVs) with the presynaptic plasma membrane at specialized release sites termed active zones (AZs) (Galli and Haucke, 2004, Kittel et al., 2006a, Murthy and De Camilli, 2003, Sudhof and Rothman, 2009). To sustain neurotransmission it is essential that SV fusion is immediately followed by the compensatory endocytic retrieval of previously exocytosed SV membranes. Compensatory SV endocytosis prevents expansion of the presynaptic plasma membrane, thereby preserving the close alignment of presynaptic and postsynaptic structures. In addition, endocytosis restores membrane tension and is a prerequisite for the reformation of SVs, which are needed to replenish the releasable SV pool (Dittman and Ryan, 2009, Kononenko and Haucke, 2015, Murthy and De Camilli, 2003, Soykan et al., 2016). Moreover, endocytic proteins may be required in a way that is poorly understood to regenerate or clear release sites from exocytosed material to enable vesicle reloading during high-frequency firing (Haucke et al., 2011, Hosoi et al., 2009, Hua et al., 2013, Neher, 2010).