Abstract 26

INTERACTION OF GTP-BINDING RAB3D WITH TCTEX-1, A LIGHT CHAIN OF THE CYTOPLASMIC DYEIN MICROTUBULE MOTOR COMPLEX

Nathan J. Pavlos¹, Jiake Xu¹, Amerigo Carrello¹, Karen Kroeger², Karin Ednet², Ming H. Zheng¹

^1. Unit of Orthopaedics, School of Surgery and Pathology, University of Western Australia, Nedlands, Western Australia
^2. Western Australia Institute for Medical Research, QEII Medical Centre, Nedlands, Western Australia

Rab3D belongs to the family of small GTPases involved in the regulation of exocytosis. Previously we have shown that Rab3D is required for the maintenance of the osteoclastic resorptive organelle, namely the ruffled border membrane. Here, to further delineate the mechanism(s) underlying this phenomenon, we have employed a yeast two-hybrid system to identify potential Rab3D interacting proteins. Screening a mouse embryonic cDNA library, 5 clones (M-18, -30, -37, -39 and -40) were positively identified which specifically interacted with the N-terminus of Rab3D. Database searches identified these clones as mouse Tctex-1, a 14 kDa light chain of the multimeric cytoplasmic dynein motor complex. A specific interaction between Rab3D and Tctex-1 was confirmed by GST-pull down and co-localisation studies. Truncation analyses mapped the Tctex-1 binding site to the switch II/GTP-binding motif of Rab3D (amino acids 74-95). Consistently, bioluminescence resonance energy transfer (BRET) analysis demonstrated that Tctex-1 preferentially associated with the GTP-bound conformation form of Rab3D in live cells. When overexpressed, Flag-Tctex-1, GFP-dynamitin, or antisense-Tctex-1 disrupted the spatial distribution of Rab3D in vivo. Additionally, Rab3Dsecretory granules localise to microtubules and are redistributed by nocodazole treatment into ß-COPI-positive Golgi mini-stacks in transfected COS-1 cells. These data lend support to the notion that Rab GTPases and molecular motor proteins act in concert to regulate directional membrane transport and furthermore suggest that Rab3D may functions to recruit and regulate the activity of cytoplasmic dynein Tctex-1, controlling the sorting and microtubuledependent targeting of post-Golgi secretory granules to the ruffled border membrane during osteoclastic bone resorption.

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