Protein targeting to endosomes/lysosomes: function of Septins in AP-3-dependent transport

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Clathrin-coats are involved in various aspects of lysosome biogenesis. Clathrin associates with hetero-tetrameric adaptor proteins (APs). While AP-2 functions in endocytosis, the ubiquitously expressed AP-1 A, AP-3 and AP-4 are required for the transport of transmembrane proteins from the secretory pathway to the endosomal/lysosomal system. Using a liposome-based in vitro systems that recapitulate the fidelity of protein sorting in vivo, we recently identified the protein networks supporting AP-1 and AP-3 sorting functions, which collectively highlight the functional importance of =80 proteins. In this proposal, we will focus on the functional characterization of the protein networks supporting AP-3 sorting function in targeting of lysosomal membrane associated proteins. In particular, we will investigate the function of several septin family members and their regulator Borg4 in this process. Our working hypothesis is that septins, by interacting with proteins of the AP-3 sorting machinery, facilitate the binding of AP-3-coated membranes onto microtubules and facilitate microtubule-dependent transport from early to late endosomes. To test this hypothesis, it will be necessary 1) to colocalize septins and AP-3, 2) to establish the functional importance of septins in membrane traffic along the endocytic pathway, in particular during the early to late endosome transition, 3) to test whether septins are found along specific microtubule tracks, 4) to establish the molecular links between septins and their effector Borg4 with other proteins belonging to the AP-3 network, and 5) to understand how septin function is regulated by Borg4, and the cdc42 GTPase and by phosphorylation.