Cytoplasmic control of Rab family small GTPases through BAG6.

Toshiki Takahashi,Yugo Tsuchiya,Shin‐Ichi Hisanaga,Kazu Tajima,Natsumi Sakai,Setsuya Minami,Norihiko Ohbayashi,Kei Suga,Mitsunori Fukuda,Hiroyuki Kawahara

doi:10.15252/embr.201846794

Abstract

Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP‐bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP‐associated Rab8a, while its major GTP‐bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP‐Rab8a via the ubiquitin‐proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6‐mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.

Highlights

Rab family small GTPases are critical for maintaining the function and architecture of cytoplasmic organelles by controlling membrane trafficking between them [1,2,3,4,5]
The transferrin receptor (TfnR), an endosomal protein that is delivered from the trans-Golgi network (TGN) to tubular recycling endosomes (RE) [14,45,46], was found scattered around a peripheral region in the cytoplasm of BAG6-suppressed HeLa cells (Fig 1A, right panel)
In the case of the 12-pass transmembrane domain (TMD) protein Patched 1 (Ptc1), it was reported that the majority of this endosomal protein can be detected in cytoplasmic vesicular structures [49], and our immunostaining in control cells showed a similar pattern of typical Ptc1 signals (Fig 1B, left panel)

Summary

Introduction

Rab family small GTPases are critical for maintaining the function and architecture of cytoplasmic organelles by controlling membrane trafficking between them [1,2,3,4,5]. Together with SNARE proteins, which mediate vesicle docking and fusion [8,9], distinct Rab GTPases localize to different membrane compartments in order to control the specificity and directionality of membrane trafficking pathways [5]. Rab proteins control membrane trafficking by cycling between active GTP-bound and inactive GDP-bound forms that differ primarily by the conformation of two nucleotide-surrounding loops known as “Switch” regions [16]. These conformational changes are critical, since Rab proteins in the GTP-bound form at the membrane become competent to bud and recruit vesicles to target destinations with specific effector interactions [17,18,19]. Several chaperone-like factors, including GDP-dissociation inhibitors (GDIs), Rab escort protein (REP) and RABIF/MSS4 (mammalian suppressor of yeast Sec4), have been reported to help prevent the aggregation of Rab proteins [22,23,25,26,27], the cytoplasmic machinery that determines the fate of the GDPbound form of specific Rab species remains largely unknown

Methods

Results

Conclusion