Resumen de Analysis of the molecular function of kazrin in endosomal trafficking
Endocytosis and endosomal trafficking are of central meaning in all eukaryotic cells. It enables cells to rapidly modify their surface exposure of signalling complexes, nutrient and ion transporters, to maintain the homeostasis of lipids and proteins, to distribute internalized material within the cell, and to initiate and maintain cell motility, among others. Hence, the endocytic uptake and transport processes can regulate the capacity of the cell to sample and react to with their environment.
This thesis focused on the study of kazrin C, a human protein originally identified in our group, whose overexpression interferes with clathrin-mediated endocytosis (CME), one of the best studied endocytic pathways. Preliminary experiments demonstrated that kazrin C interacts with the clathrinassociated machinery from rat brain extracts. Further, that data indicated that depletion of kazrin accelerates CME and recycling of the widely used endocytic marker transferrin (Tfn), whereas it impairs arrival of internalized Tfn to the perinuclear endosomal recycling compartment (ERC). Last, initial experiments suggested that kazrin might localized to sorting endosomes (SE), together with rab4, γ-adaptin and EHD3.
The work presented here confirmed the previously observed effects in Tfn uptake and endocytic trafficking upon kazrin depletion. Further, subcellular fractionation experiments biochemically reinforced the view that a fraction of kazrin is localized to endosomal compartments. Consistently also, transfected GFP-kazrin could be seen forming subdomains on YFP-rab4 positive endosomes in living cells. Further, we provide biochemical evidence indicating that kazrin C directly interacts with clathrin and the clathrin adaptor AP-1, which together with rab4b, have been implicated in the transport between the sorting and recycling endosomes, similar to kazrin. Finally, we found that kazrin co-localize with endosomal actin and that it directly interacts with the Arp2/3 complex and WASH, the main the activator of the Arp2/3 complex on endosomal membranes. Both, depletion and overexpression of kazrin resulted in accumulation of endosomal actin. The data suggest that kazrin might inhibit cytosolic WASH, but upon activation, it might induce WASH-dependent actin polymerization on AP-1 and clathrin-enriched endosomal subdomains, promoting the formation or fission of transport intermediates between the sorting and recycling compartments.
Altogether, the results indicated a role for kazrin at the sorting endosomal recycling trafficking crossroad, favouring transport towards the ERC together with clathrin and AP-1, versus the short loop recycling path to the plasma membrane. At the molecular level, the data suggest kazrin might control the balance of WASH-induced actin polymerization on either retromer or clathrin-enriched endosomal subdomains.
