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Drosophila Rolling Blackout Displays Lipase Domain‐Dependent and ‐Independent Endocytic Functions Downstream of Dynamin
Author(s) -
Vijayakrishnan Niranjana,
Phillips Scott E.,
Broadie Kendal
Publication year - 2010
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2010.01117.x
Subject(s) - dynamin , biology , microbiology and biotechnology , endocytosis , endocytic cycle , lipase , mutant , gtpase , biochemistry , receptor , gene , enzyme
Drosophila temperature‐sensitive rolling blackout ( rbo ts ) mutants display a total block of endocytosis in non‐neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G‐X‐S‐X‐G active site, and assayed genomic rescue of rbo mutant non‐neuronal and neuronal phenotypes. The rbo S358A mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase domain is critical for RBO essential function. Likewise, the rbo S358A mutant cannot provide any rescue of endocytic blockade in rbo ts Garland cells, showing that the lipase domain is indispensable for non‐neuronal endocytosis. In contrast, rbo ts conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rbo S358A mutant, showing that the RBO lipase domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbo ts and the well‐characterized dynamin GTPase conditional shibire ( shi ts1 ) mutant. In both non‐neuronal cells and neuronal synapses, shi ts1 ; rbo ts phenocopies shi ts1 endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase domain‐dependent and scaffolding functions with differential requirements in non‐neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity.