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

TY - JOUR

T1 - Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy

AU - Gavriljuk, Konstantin

AU - Itzen, Aymelt

AU - Goody, Roger S

AU - Gerwert, Klaus

AU - Kötting, Carsten

PY - 2013/8/13

Y1 - 2013/8/13

N2 - 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 membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.

AB - 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 membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.

KW - Animals

KW - Cattle

KW - Guanine Nucleotide Dissociation Inhibitors

KW - Kinetics

KW - Legionella pneumophila

KW - Membranes

KW - Phosphorylcholine

KW - Prenylation

KW - Saccharomyces cerevisiae

KW - Spectrophotometry, Infrared

KW - Spectroscopy, Fourier Transform Infrared

KW - Transport Vesicles

KW - rab GTP-Binding Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1073/pnas.1307655110

DO - 10.1073/pnas.1307655110

M3 - SCORING: Journal article

C2 - 23898197

VL - 110

SP - 13380

EP - 13385

JO - P NATL ACAD SCI USA

JF - P NATL ACAD SCI USA

SN - 0027-8424

IS - 33

ER -