TAT-1, a phosphatidylserine flippase, affects molting and regulates membrane trafficking in the epidermis of Caenorhabditis elegans.

Abstract

Membrane trafficking is a conserved process required for the import, export, movement, and distribution of proteins and other macromolecules within cells. The Caenorhabditis elegans NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking and are required for the completion of molting. Using a genetic approach, we identified reduction-of-function mutations in tat-1 that suppress nekl-associated molting defects. tat-1 encodes the C. elegans ortholog of mammalian ATP8A1/2, a phosphatidylserine flippase that promotes the asymmetric distribution of phosphatidylserine on the cytosolic leaflet of lipid membrane bilayers. CHAT-1 (human CDC50), a conserved chaperone, was required for the correct localization of TAT-1, and chat-1 inhibition strongly suppressed nekl defects. Using a phosphatidylserine sensor, we found that TAT-1 was required for the normal localization of phosphatidylserine at apical endosomes and that loss of TAT-1 led to aberrant endosomal morphologies. Consistent with these data, TAT-1 localized to early endosomes and to recycling endosomes marked with RME-1, the C. elegans ortholog of the human EPS15 homology domain-containing protein, EHD1. TAT-1, phosphatidylserine biosynthesis, and the phosphatidylserine-binding protein RFIP-2 (human RAB11-FIP2) were all required for the normal localization of RME-1 to apical endosomes. Consistent with these proteins functioning together, inhibition of RFIP-2 or RME-1 led to the partial suppression of nekl molting defects, as did inhibition of phosphatidylserine biosynthesis. We propose that TAT-1 flippase activity, in conjunction with RFIP-2, promotes the recruitment of RME-1 to apical recycling endosomes and that inhibition of TAT-1-RFIP-2-RME-1 can compensate for a reduction in NEKL activities.