Abstract
Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.
Highlights
DGOLPH3-RFP and its associated partners were characterized by RFP affinity purification, coupled with mass spectrometry (AP-MS), and selected interactors were subsequently validated by co-immunoprecipitation (Co-IP) or gluthatione S-transferase (GST) pull-down
We have exploited the advantages of Drosophila spermatogenesis, which offers a well-suited model system for dissecting membrane trafficking pathways and their role in cytokinesis and cell differentiation [23,24,25,26,27]
Consistent with our previous findings that Drosophila GOLPH3 (dGOLPH3) controls membrane trafficking during cytokinesis [4,21,22,27], the dGOLPH3 interactome revealed an enrichment of proteins involved in cytokinesis and vesicle-mediated trafficking
Summary
4-phosphate [PI(4)P] binding protein, required for maintenance of Golgi structures and protein trafficking [1]. GOLPH3 function has been involved in multiple vesicular routes including vesicular transport to the plasma membrane and intra-Golgi and endocytic trafficking [1,2,3,4,5,6]. Many studies revealed that GOLPH3 is required for coatomer (COPI)mediated Golgi trafficking of several protein glycosyltransferases [7,8,9,10,11,12,13]. All Golgi glycosyltransferases are type II membrane proteins containing a small cytosolic N-terminal region, a single transmembrane domain and a luminal enzymatic domain [14]. Vps74p, the yeast homolog of GOLPH3, binds to the COPI coatomer as well as to a (F/L)(L/I/V)XX(R/K)
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