Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila

Tricellular junctions (TCJs) provide essential adhesive and occluding functions at epithelial cell vertices and play key roles for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka), Gliotactin (Gli), and M6 constitute tricellular occluding junctions. Aka and M6 localize in an interdependent manner to vertices and are required jointly to localize Gli, but how these proteins interact to assemble TCJs was not known. Here, we show that the tetraspan proteolipid protein M6 physically interacts with Aka and with itself. M6 is palmitoylated on a conserved juxta-membrane cysteine cluster. This modification promotes efficient vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed accumulation of M6 and Aka at vertices but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, while subsequent TCJ growth relies on different mechanisms independent of M6 palmitoylation.