Function of the trimeric Rab7 GEF complex in endolysosomal biogenesis in Drosophila

• Applicants: Professor Dr. Achim Paululat; Professor Dr. Christian Ungermann
• Subject Area: Cell Biology ; Animal Physiology and Biochemistry
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 248987912

The endolysosomal system connects the plasma membrane via endosomal uptake to the lysosomal protein degradation and recycling machinery of the cell. Thereby, the endolysosomal pathway controls both the integrity of its limiting membrane and functionality of the embedded membrane proteins, but also cellular signaling cascades. Endosomes mature from early to late endosomes, which requires a coordinated exchange of the early endosomal Rab5 by the late endosomal Rab7 GTPase as a prerequisite of late endosome fusion with the lysosome. Our data revealed that the trimeric Drosophila Mon1-Ccz1-Bulli complex does not requires Bulli for its Rab7 GEF activity or Rab5-dependent activation. However, loss of Bulli causes a massive accumulation of Rab5 at late endosomes that causes physiological deficits in bulli mutant animals. Our data suggest a role of Bulli in endosomal maturation by either inhibiting the Rab5 GEF or by recruiting the Rab5 GAP resulting in both cases in an accumulation of membrane-bound hyperactive Rab5. Within this proposal we will take a combined in vivo and in vitro approach to dissect the function of the Drosophila Rab7 GEF complex in detail. Within Aim 1, we will determine the molecular environment of the Rab7 GEF complex by searching for specific interactions in Drosophila and insect cells using improved Bulli protein purification and next-neighbor crosslinking followed by mass spectrometry. Possible candidates and sites will be evaluated in Aim 2 and 3. In Aim 2, we will focus on the in vivo function of Bulli by analyzing the consequences of Rab5 GAP or GEF deletions or overexpression on Rab5 and Rab7 localization, both in wild-type and bulli mutant animals, and will clarify the role of novel interactors. In Aim 3, we will take advantage of our in vitro system (i) to recapitulate if we find any specific role of Bulli within the Rab7 GEF complex in Rab5-dependent Rab7 activation, (ii) determine the role of the Rab7 GEF complex on the catalytic activity of the Rab5 GAP or GEF, and (iii) determine the role of new interactors and posttranslational modifications in GEF and GAP assays. We finally aim to reconstitute a possible circuit between Rab5 inactivation and Rab7 activation using supported lipid membranes to determine how this Rab cascade is regulated in detail. We expect that this analysis will clarify the coordination of EE to LE and lysosome maturation in metazoan cells as a prerequisite for possible interventions in biomedicine.

• DFG Programme: Research Grants