Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange

Feng

et al. 1998

MBoC

350

293

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum–to–Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Section: Rab11 Is Selectively Released From Membranes By Overexpressementioning

confidence: 99%

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

Feng

et al. 1998

MBoC

350

293

European Journal of Biochemistry

“…The chloroform phase was collected and the sample re-extracted with the same volume of chloroform. The combined chloroform extract was evaporated and 30 ml 25 mm NH 4 OH added. The presence of geranylgeranyl alcohol was determined by electrospray ionization MS using an LCQ mass spectrometer (Finnigan, San Jose, CA, USA).…”

Section: Expression and Purification Of Tev Proteasementioning

confidence: 99%

“…For their function Rab proteins require modification by geranylgeranyl isoprenoids. This allows them to associate reversibly with a membrane in a tightly controlled fashion [3,4]. Covalent attachment of geranylgeranyl groups to two C-terminal cysteines is catalyzed by Rab geranylgeranyl transferase (RabGGTase).…”

mentioning

confidence: 99%

“…Two known mammalian proteins, REP-1 and REP-2, share 75% amino acid sequence identity and functionally overlap [14]. Both of them have several stretches of high sequence homology to Rab-GDP dissociation inhibitor, a cytosolic factor responsible for delivery and removal of Rab proteins to and from the membrane [3,4,15]. According to the current model, a newly synthesized Rab protein forms a stable complex with REP [7,16].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase

Alexandrov

Simón²,

Yurchenko³

et al. 1999

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7±REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7±REP complex. For this interaction we determined a K d value of about 120 nm.Association of RabGGTase with the Rab7±REP complex occurs with a rate constant of < 10 8 m 21´s21 . We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7±REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7±REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then`scans' the flexible C-terminus to position the proximal cysteines into the active site.Keywords: geranylgeranylation; prenyltransferase; Rab7; Rab geranylgeranyl transferase; REP.Rab proteins are members of the Ras superfamily of small GTP-binding proteins. Many of them were shown to be implicated in control of intracellular vesicular traffic [1,2]. For their function Rab proteins require modification by geranylgeranyl isoprenoids. This allows them to associate reversibly with a membrane in a tightly controlled fashion [3,4]. Covalent attachment of geranylgeranyl groups to two C-terminal cysteines is catalyzed by Rab geranylgeranyl transferase (RabGGTase).Mammalian RabGGTase (GGTase II) is a heterodimer composed of tightly associated a and b subunits of 60 and 38 kDa, respectively [5,6], and belongs to the family of protein prenyl transferases together with farnesyl transferase and geranylgeranyl transferase I (GGTase I) [7]. Substrates of farnesyl transferase include members of the Ras family, lamin b and...

“…Early studies suggested the existence of GDF, a factor that would facilitate delivery of Rab-GDP to membranes from the cytosolic GDI-Rab complex (22,26). For example, GDF could be a Rab guanine nucleotide exchange factor(s) (GEF) that promotes Rab activation to the GTPbound form.…”

mentioning

confidence: 99%

A New Functional Domain of Guanine Nucleotide Dissociation Inhibitor (α‐GDI) Involved in Rab Recycling

Luan

Heine

Zeng

et al. 2000

Traffic

Guanine nucleotide dissociation inhibitor (GDI) is a 55-kDa protein that functions in vesicular membrane transport to recycle Rab GTPases. We have now determined the crystal structure of bovine a-GDI at ultra-high resolution (1.04 A , ). Refinement at this resolution highlighted a region with high mobility of its main-chain residues. This corresponded to a surface loop in the primarily a-helical domain II at the base of a-GDI containing the previously uncharacterized sequence-conserved region (SCR) 3A. Site-directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane-bound Rab. This domain, referred to as the mobile effector loop, in combination with Rab-binding residues found in the multi-sheet domain I at the apex of a-GDI may provide flexibility for recycling of diverse Rab GTPases. We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.