Functional Characterizations Of P4 ATPase TAT-1 And The Chaperone CHAT-1 In Regulating Endocytic Sorting And Recycling In C. Elegans

AbstractIn eukaryotic cells, internalized cargoes are transported to early endosomes where they are sorted to be recycled back to the plasma membrane, degraded in lysosomes or delivered to the trans-Golgi network. Early endosomes display a complex and pleiomorphic organization with many tubular processes emanating from central vesicular elements. Receptors and other membrane proteins that are to be recycled concentrate in the tubular extensions. The recycling vesicles which arise from the tubular extensions may undergo fast recycling, by fusing directly with plasma membranes, or slow recycling, by transporting cargoes through the endocytic recycling compartment (ERC). Both cargo sorting and subsequent recycling require extensive membrane remodeling to form tubular extensions. However, it is not clear at present how these tubular processes are formed and maintained.From a genetic screen for regulators of phosphatidylserine (PS) signal, we isolated several alleles of tat-1 (transbilayer amphipath transporters) and an allele of chat-1 (chaperon of tat-1) which encodes a Cdc50 family protein. tat-1 encodes a P4-type ATPase that is suggested to promote the inward movement of aminophospholipids such as PS and PE, thereby restricting them to the inner leaflet of cell membranes. Consistent with previous findings, we found that in the tat-1 mutants, the surface-exposed PS is detected not only on apoptotic cells, but also on living cells. Identical phenotypes were also observed in the chat-1(qx36) mutants, indicating that chat-1 is also involved in maintaining PS asymmetry on plasma membranes. TAT-1 and CHAT-1 are mutually required for exiting the ER, similar to P4-ATPase and Cdc50p proteins in yeast and mammalian cells.In chat-1 and tat-1 mutants, the endocytic sorting process is disrupted, leading to defects in both cargo recycling and degradation. TAT-1 and CHAT-1 are expressed in the same tissues and colocalize to both plasma membranes and intracellular compartments, including the tubular domain of the early endosome and the recycling endosome where PS is enriched on the cytosolic surface. Loss of tat-1 and chat-1 function disrupts membrane PS asymmetry and abrogates the tubular membrane structure. Furthermore, surface charge and protein targeting are altered in tat-1 and chat-1 mutants. Our data suggest that CHAT-1 and TAT-1 maintain membrane phosphatidylserine asymmetry, and thus regulate endocytic sorting and recycling by promoting membrane tubulation. In addition, restriction of PS on the cytosolic leaflet by TAT-1 and CHAT-1 may also mediate the targeting of positively charged and/or PS-associated regulatory proteins, thereby promoting endocytic transport.In order to identify factors which may function redundantly with TAT-1, we performed a forward genetic screen to search for mutants which are synthetically lethal with tat-1 (If). Up to now,5 mutants were isolated and mapped to different chromosomes. We are in the process of further mapping and cloning of the genes affected in these mutants.