In eukaryotic cells, endocytic cargoes are transported to early endosomes where they are sorted to be recycled back to plasma membranes, degraded in lysosomes or delivered to trans-Golgi network.
tat-1, which encodes a C. elegans P4-ATPase, is required for endocytic sorting and recycling by maintaining membrane phosphatidylserine (PS) asymmetry1.
tat-1(lf) mutants accumulate big intestinal vacuoles and display various defects in endocytic transport 1,2. To identify more regulators involved in this process, we performed a genetic screen to search for mutants which suppress the vacuolation phenotype in the intestine of
tat-1(
qx30) animals. From a screen of 12,000 haploid genomes, we have isolated two recessive mutations
qx49 and
qx71, which completely suppress the appearance of abnormal vacuoles in the intestine of
tat-1(
qx30).
qx49 and
qx71 also suppress the vacuolation phenotype in animals lacking CHAT-1, the chaperon of TAT-1. By examining different steps of endocytic transport, we found that both
qx49 and
qx71 mutations efficiently reversed the cargo recycling defects in
tat-1(
qx30) mutants. In addition, the abnormally aggregated RAB-5-, RAB-7-, RAB-10- and RME-1-positive vesicles in the
tat-1(
qx30) intestine appeared to be normal in
qx49qx30 double mutants. We found that membrane PS asymmetry is still defective in
qx49qx30 and
qx30;
qx71 worms, which indicates that the suppression of defective endocytic transport in the double mutants is not due to a correction of PS distribution. We have cloned the gene affected in
qx49 and
qx71 and are in the process of characterizing their functions in endocytic transport. We will report our progress in the meeting. References: 1. Chen B, Jiang Y, Zeng S, Yan J, Li X, et al. (2010) Endocytic Sorting and Recycling Require Membrane Phosphatidylserine Asymmetry Maintained by TAT-1/CHAT-1. PLoS Genet 6(12):
e1001235. doi:10.1371/journal.pgen.1001235. 2. Anne-Francoise Ruaud, Lars Nilssoon, Fabrice Richard, Morten Krog Larsen, Jen-Louis Bessereau and Simon Tuck, 2009. The C. elegans P4-ATPase TAT-1 regulates lysosome biogenesis and endocytosis. Traffic 10(1): 88-100.