Rab7 CMT2B Mutants Alter function in Endosomal Transport, Growth Factor Signaling and Regulatory Protein Interaction

Abstract

Rab7 regulates transport and degradation on the late endocytic pathway by orchestrating interactions with motor proteins, proteasome, lipid kinases and phosphatases. Four different point mutants of Rab7 have been identified as a causative trigger for Charcot‐Marie‐Tooth Type 2B disease (CMT2B), inherited neuropathies affecting the peripheral nerves. Thus, CMT2B neuropathies are characterized by sensory loss, muscle weakness often leading to infections and consequently amputations. Disease causing mutations in Rab7 alter amino acid residues in the GTP‐binding and hydrolysis domains of Rab7. Through biochemical and morphologic assays we find the mutant GTPases exhibit altered functionality due to disrupted protein‐protein interactions and growth factor signaling. CMT2B mutants also exhibit increased GTP‐binding, and slowed membrane cycling, parameters that depend on regulatory guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) interactions. We have established that Vps39 functions as a Rab7 GEF and TBC domain family, member 15 (TBC1D15) is reported to serve as a Rab7 GAP. Therefore, we comparatively evaluated wild‐type Rab7 and the four known CMT2B mutants (L129F, K157N, N161T, and V162M) in GEF and GAP assays. Regulatory protein facilitated nucleotide exchange and hydrolysis by N161T was comparable to Rab7 wild‐type. The remaining mutants were comparable to the constitutively active Rab7Q67L mutant. Thus, CMT2B neuropathy results from an interference with both Rab7 function and nucleotide activation status. NSF MCB‐0446179 (AWN) and NIH 2R25GM075149‐04 (PJ) grants support work.