Abstract
Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division.
Highlights
Phosphatidylinositol (PI)3 is a relatively minor component of lipid membranes in most cells, making up ϳ8% of total phospholipid
Class III PI 3-kinases have been implicated in a variety of processes, one of the best understood being their role as downstream effectors of the small GTPase, Rab5, in endosomal membrane fusion
Primary structural analysis places the TbVps34 gene product as a Class III PI 3-kinase, and based on the effects following knockdown by RNA interference (RNAi), TbVps34 clearly shares with its mammalian and S. cerevisiae orthologues a central role in vesicular trafficking and endocytosis
Summary
Phosphatidylinositol (PI) is a relatively minor component of lipid membranes in most cells, making up ϳ8% of total phospholipid. Yeast Vps34p regulates transport to the vacuole, whereas the human class III PI 3-kinase, hVPS34, is required for multivesicular body morphogenesis and for transport of lysosomal proteins (9 –11). Class III PI 3-kinases have been implicated in a variety of processes, one of the best understood being their role as downstream effectors of the small GTPase, Rab, in endosomal membrane fusion. Class III PI 3-kinases are required for vacuolar transport in yeast and multivesicular body formation in mammals but have been implicated in the processes of retrograde endosome to trans-Golgi network (TGN) trafficking, via direct binding to retromer, and in autophagy [3, 20, 21]. We describe the identification and functional analysis of TbVps, the trypanosome orthologue of Vsp; in particular we uncovered an unexpected role for TbVps in segregation of the Golgi apparatus at cytokinesis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
