Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases to the endosomal pathway- they then return to the Golgi apparatus in a process mediated by the GTPase Rab9 and its effectors. Work in the second chapter of this thesis describesMoreMannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases to the endosomal pathway- they then return to the Golgi apparatus in a process mediated by the GTPase Rab9 and its effectors.
Work in the second chapter of this thesis describes investigations involving the putative tether and Rab9 effector GCC185 and its role in MPR trafficking. A yeast two hybrid screen in conjunction with direct biochemical assays was used to identify Rab proteins that bind to GCC185 along its length. One of these Rab binding sites, within a central coiled coil domain, was shown to be important for trafficking of MPRs to the Golgi apparatus.
We also demonstrated that full length GCC185 is necessary for GCC185 function, and showed that a GCC185 construct lacking N-terminal portions is unable to rescue phenotypes in cells depleted of GCC185. Binding of multiple Rabs along the length of GCC185 may assist in stacking interactions of Golgi cisternae and participate in functions related to its role as a vesicle tether.-In chapter three, we examine the role of motor proteins in MPR transport.
We identified a kinesin-like protein known as KIF1C as a direct Rab9 binding partner and characterized its motor properties. The protein was shown to be a plus-end directed motor that moves microtubules in vitro at a speed comparable to that of late endosomes in vivo. We further demonstrated that this motor interacts with Rab9 in a nucleotide dependent fashion and was able to relocalize an activated Rab9 construct, Rab9Q66L, upon coexpression in living cells. The interaction of Rab9 directly with a motor protein suggests models for vesicle movement that contribute to correct recycling of MPRs.-The fourth chapter describes the extensive characterization of the Rab9 effector p40 and assesses its role in MPR trafficking.
We demonstrated that cells lacking p40 contain incorrectly localized MPRs into non-classical endosomal structures. Lack of p40 has substantial consequences on MPR trafficking in vivo, as demonstrated by their slowed return to the Golgi complex and hypersecretion of hexosaminidase.
Furthermore, p40 is also shown to bind directly to the gamma ear domain of the AP-1 clathrin adaptor, a protein also mislocalized upon depletion of p40. These experiments suggest a model in which p40 acts in the process of collecting MPRs into late endosomal compartments prior to return to the Golgi apparatus.