The processing, sorting and signaling functions that take place at the Golgi apparatus are widely appreciated to be significant in health and disease. Therefore, it is important to identify the components that confer the functional organization of the Golgi apparatus. Golgins- a set of elongated Golgi proteins that are prominent autoantigens in a wide-array of autoimmune disorders - are strong candidates, but their actual roles remain unknown. To identify proteins required for Golgi structure and function a directed siRNA-based screen targeting candidates, especially golgins, is being used with cell-based Golgi assembly and trafficking assays. As a result, we are defining conceptually discrete steps in Golgi assembly and the requirements at these steps. Further, by analyzing cells with Golgi assembly blocked at a given step, we are identifying the importance of Golgi organizational features such as compartmenalization, positioning, and ribbon formation. The focus of this proposal is on the golgins p115 and GM130 which we identified as requirements for Golgi compartmentalization and ribbon formation, respectively. Gene replacement in conjunction with permeabilized cell assays and live imaging will be used to analyze p115 and GM130 structure/function relationships. To date, we have identified two required p115 domains: the SNARE-binding cc1 domain and, in unpublished work, the conserved homology region 1 (HR1) domain. Significantly, HR1 binds the COPI vesicle coat and a point mutation abolishing this interaction disrupts p115 activity upon gene replacement. These results set the stage for tests of p115/COPI interactions in COPI vesicle docking and of p115/SNARE interactions in SNARE pairing. For GM130, we have shown that it requires its GRASP65-binding domain, that GRASP65 itself is required for Golgi ribbon formation, and that in the absence of the ribbon, Golgi enzymes fail to equilibrate causing defective processing. Next, we will test the hypothesis that after recruitment to the Golgi by GM130, GRASP65 oligomerization on adjacent ministacks drives SNARE- mediated membrane fusion to form the ribbon. Remarkably, our work on Golgi ribbon forming complexes is also elucidating a novel cell cycle control mechanism. We have shown that the GRASP family member GRASP55 is mitotically phosphorylated by the MEK/ERK pathway and, in unpublished work, that GRASP55 is also required for linking Golgi ministacks into ribbons. Further, MEK inhibition delays the G2/M cell cycle transition and this is suppressed by prior unlinking of the Golgi. Thus, we will test the hypothesis that MEK/ERK phosphorylation of GRASP55 in late G2 unlinks the Golgi promoting CDK1 activation and G2/M.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cell Structure and Function (CSF)
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Ainsztein, Alexandra M
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Carnegie-Mellon University
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Song, Lina; Bachert, Collin; Linstedt, Adam D (2016) Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo. Methods Mol Biol 1496:123-31
Song, Lina; Bachert, Collin; Schjoldager, Katrine T et al. (2014) Development of isoform-specific sensors of polypeptide GalNAc-transferase activity. J Biol Chem 289:30556-66
Jarvela, Timothy S; Linstedt, Adam D (2014) The application of KillerRed for acute protein inactivation in living cells. Curr Protoc Cytom 69:12.35.1-12.35.10
Tewari, Ritika; Jarvela, Timothy; Linstedt, Adam D (2014) Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin. Mol Biol Cell 25:3049-58
Mukhopadhyay, Somshuvra; Linstedt, Adam D (2013) Retrograde trafficking of AB? toxins: mechanisms to therapeutics. J Mol Med (Berl) 91:1131-41
Guo, Yusong; Linstedt, Adam D (2013) Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat. Cell Logist 3:e27687
Mukhopadhyay, Somshuvra; Redler, Brendan; Linstedt, Adam D (2013) Shiga toxin-binding site for host cell receptor GPP130 reveals unexpected divergence in toxin-trafficking mechanisms. Mol Biol Cell 24:2311-8
Bachert, Collin; Linstedt, Adam D (2013) A sensor of protein O-glycosylation based on sequential processing in the Golgi apparatus. Traffic 14:47-56
Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D et al. (2012) Genetically encoded pH sensor for tracking surface proteins through endocytosis. Angew Chem Int Ed Engl 51:4838-42
Jarvela, Timothy; Linstedt, Adam D (2012) Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery. J Cell Sci 125:973-80

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