Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy

Abstract

Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection-Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection-Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membranebound Rab GTPases and, thus, is not merely a solubilization factor. lipid bilayers | vesicle spreading | Rab recycling | geranylgeranyl

Rab35 GTPase: A Central Regulator of Phosphoinositides and F‐actin in Endocytic Recycling and Beyond

“…Consequently, AnkX expression reduces Rab35 binding to its GEF DENND1A and mimics Rab35 depletion. In addition, phosphocholination reduces the extraction of Rab35 from membranes by GDI (GDP dissociation inhibitor), at least in vitro . Besides AnkX, Legionella secrete another effector protein (LepB) that in vitro displays GAP activities toward Rab1, Rab3, Rab8, Rab13 and Rab35 .…”

Rabs on the fly: Functions of Rab GTPases during development

“…Recently, one study using attenuated total reflectionFourier transform infrared (ATR-FTIR) spectroscopy proposed that GDP-dissociation factors (GDI) [20][21][22] directly mediate membrane extraction. 23 ATR-FTIR involves the collection of infrared radiation reflected from the interphase between an aqueous phase and a prism, in which the evanescent wave penetrated from the prism into the aqueous solution is absorbed by substances solved in the aqueous phase. 24 The subsequent formation of cytosolic Rab-GDP/GDI complexes enables inactive Rab-GDPs to recycle and reintegrate into their original membranes.…”

Rab Proteins and the Compartmentalization of the Endosomal System

“…Second, Rab GTPases behave both as soluble and specifically localized, integral-membrane proteins. Rab GTPases are kept soluble in the cytosol and in the inactive (GDP-bound) conformation through association with Rab GDP-dissociation inhibitors (Rab-GDI) (Ullrich et al 1993;Gavriljuk et al 2013). Prenylation-addition of one or two geranyl-geranyl groups-on conserved carboxyterminal cysteine residues serves together with upstream hypervariable regions in promoting specific and stable membrane association (Sa-saki et al 1990;Chavrier et al 1991;Seabra et al 1991;Kinsella and Maltese 1992;Ullrich et al 1993;Seabra and Wasmeier 2004;Lane and Beese 2006;Wu et al 2010).…”