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M Wessling-Resnick

Publications and source records attributed to M Wessling-Resnick.

At least 37 records · Page 2Linked to original sources
PubMed1996

Influence of Mg2+ on the structure and function of Rab5.

Mg2+ inhibits GDP release from Rab5WT but not from Rab5S34N, a mutant lacking Ser34 critical for Mg2+ coordination in the nucleotide binding pocket. Thus, inhibition of GDP release is apparently exerted via coordination of Mg2+ between Rab5 and GDP. Mg2+ also induces conformational changes in Rab5WT, demonstrated by increased tryptophan fluorescence intensity and a red shift in lambda max for the GDP-bound protein. Mg(2+)-induced fluorescence changes are not observed for Rab5S34N. The correlation between Mg2+ effects on nucleotide exchange and the fluorescence properties of Rab5 suggests that a conformation promoted through Mg2+ coordination with Ser34 also contributes to inhibition of GDP release. The role of structural changes in GDP release was investigated using C- and N-terminal truncation mutants. Similar to Rab5WT, Mg2+ inhibits GDP release and alters the fluorescence of Rab5(1-198) but only partially inhibits release from Rab5(23-198) and fails to induce changes in the latter's fluorescence properties. Since Rab5(23-198) maintains Ser34 necessary for Mg2+ coordination, the lack of Mg(2+)-induced fluorescence changes suggests a requirement for the N-terminal domain to promote a conformation blocking GDP release. A model for mechanisms of interaction between Ras-like proteins and their exchange factors is proposed.

Amino Acid Sequence↗
PubMed1996

Fe-nitrilotriacetic acid--binding proteins associated with rat liver plasma membranes.

The uptake of nontransferrin-bound iron by hepatocytes is known to occur and may contribute to the deposition of iron and resulting injury during hemochromatosis. To examine the proteins that may function in the transport of nontransferrin-bound iron, the properties of FeNTA-binding to rat liver basolateral plasma membranes were characterized. The binding of 55FeNTA to purified liver basolateral plasma membranes was measured using a simple centrifugation assay. The binding activity could be solubilized with 0.1% octylglucoside; apparent molecular weight Mapp approximately 210 kd for the binding complex was determined by gel filtration chromatography. Immobilized metal affinity chromatography was used to further purify binding protein(s) from rat liver plasma membranes and at least six polypeptides were identified by silver staining. If associated in a stoichiometric complex, the molecular mass of these proteins would predict a size of approximately 227 kd in fairly close agreement with the gel filtration experiments. The characterization of FeNTA-binding proteins associated with basolateral membranes is the first step towards understanding elements responsible for the uptake of nontransferrin-bound iron by the liver.

Animals↗
PubMed1996

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Androstadienes↗
PubMed1996

Molecular mechanisms of iron transport.

The homeostasis of iron transport and absorption represents a critical mechanism to maintain the necessary physiologic balance of this essential nutrient. Recent advances in our understanding of the molecular mechanisms involved in the transport of iron and its membrane translocation are reviewed. Translational and transcriptional control mechanisms that have been characterized to play a role in iron homeostasis are also discussed.

Animals↗
PubMed1995

GDP dissociation inhibitor serves as a cytosolic acceptor for newly synthesized and prenylated Rab5.

In vitro synthesis and post-translational prenylation of Rab5 is accomplished using reticulocyte lysate supplemented with prenyl precursors (Sanford, J. C., Pan, Y., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 23773-23776). When Rab5 is translated in the presence of biotin-lysine-tRNA, it incorporates biotin-lysine into its peptide backbone and is efficiently prenylated; since this modification is dependent on guanine nucleotide binding, biotin-Rab5's functional integrity must be maintained. Prenylated biotin-Rab5 associates with a 45-kDa reticulocyte GDP dissociation inhibitor (GDI), sedimenting as a approximately 70-kDa particle on 5-20% sucrose density gradients. The GDI-Rab5 complex can be captured using streptavidin-linked agarose beads. Only Rab5 peptides that are substrates for prenylation are found to cosediment with the lysate GDI on sucrose gradients. Post-translational association of Rab5 and GDI is a novel finding, since previous reports suggested Rab5 remains associated with Rab escort protein (REP) after prenylation (Alexandrov, K., Horiuchi, H., Steele-Mortimer, O., Seabra, M. C., and Zerial, M. (1994) EMBO J. 13, 5262-5273). Since post-translational prenylation is catalytically mediated by REP, our study suggests that a complex between Rab5 and this factor is transient in nature. Thus, newly synthesized and prenylated Rab5 is most likely escorted to its target membrane by a GDI acceptor molecule. Biotin-Rab5 provides a novel tool for future efforts to capture and characterize additional accessory factors required for Rab protein function in vesicle transport.

Animals↗
PubMed1995

Effect of guanine nucleotide binding on the intrinsic tryptophan fluorescence properties of Rab5.

To gain further insight into structural elements involved in Rab5 function, differences in the intrinsic tryptophan fluorescence of the GDP- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound forms of the protein were examined. When excited at 290 nm, Rab5 displays emission maxima at 339.7 nm for the GDP-bound and 336.7 nm for the GTP gamma S-bound forms. The tryptophan fluorescence intensity is quenched by approximately 25% in the GTP gamma S-bound form relative to the GDP-bound conformation. Variant Rab5 molecules were created by site-directed mutagenesis to convert the protein's two tryptophans to phenylalanine residues. Fluorescence studies reveal that the observed changes upon GDP/GTP gamma S exchange are due to a blue shift in the emission spectra for both Trp74 (342.0 to 339.5 nm) and Trp114 (335.3 to 333.7 nm) and fluorescence quenching of Trp114. Consistent with the blue shift in the emission spectra, both tryptophans are more resistant to oxidation by N-bromosuccinimide in the GTP gamma S-bound state. These data indicate that both of Rab5's tryptophans are brought into a more sequestered, hydrophobic environment upon conformational changes promoted by guanine nucleotide exchange. Since Trp74 lies adjacent to Rab5's cognate switch II domain, local conformational changes would be predicted based on the known structure of Ras. However, Trp114 lies within a region of Rab5 potentially related to the switch III domain unique to heterotrimeric G alpha t. Thus, changes in the fluorescence properties of Trp114 upon guanine nucleotide exchange suggest that Rab proteins may have structure-function relationships similar to those described for heterotrimeric GTP-binding proteins.

Amino Acid Sequence↗
PubMed1995

Functional and structural interactions of the Rab5 D136N mutant with xanthine nucleotides.

Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D136N mutant of Rab5, which was predicted to switch specificity from guanine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dialysis against buffer containing XDP. The purified protein bound xanthine nucleotides with considerably higher affinity than guanine nucleotides. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant proteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in systems in which multiple GTPases are active, although interactions with non-cognate nucleotides should be evaluated if they are present at high concentration.

Base Sequence↗
PubMed1995

Phorbol esters stimulate non-transferrin iron uptake by K562 cells.

Characterization of non-transferrin (non-Tf) iron transport by K562 cells has revealed unique properties relative to iron uptake mechanisms present in other cell types (Inman, R. S., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528). Since treatment of K562 cells with phorbol esters promotes stable megakaryocytic differentiation, we examined the uptake of non-Tf iron in response to protein kinase C activation. Treatment of K562 cells with phorbol esters increased the cellular uptake of 55Fe 4-6-fold compared with untreated cells. The phorbol ester-induced stimulation of 55Fe uptake was time- and dose-dependent, with significantly enhanced transport observed only after prolonged administration of 50 nM phorbol 12,13-dibutyrate (> 8 h). These effects can be attributed to an increased Vmax of transport (14.0 +/- 5 versus 0.6 +/- 0.2 pmol/min/10(6) cells) as well as a 6-fold increase in the apparent Km (1.2 +/- 0.4 versus 0.2 +/- 0.06 microM). It is thought that the reduction of Fe3+ to Fe2+ is required as a first step in the uptake mechanism, and the associated ferrireductase activity of K562 cells is also enhanced with phorbol ester treatment by 5-10-fold (337 +/- 53 versus 43 +/- 3 pmol/min/10(6) cells). Bryostatin-1, a protein kinase C activator that fails to induce differentiation of K562 cells, did not promote this effect, indicating that the enhanced transport activity is dependent on the differentiation response. The idea that synthesis of a new class of transporters is responsible for this effect is supported by the observation that actinomycin D blocks up-regulation of non-Tf iron transport. The increased transport and ferrireductase activity induced upon differentiation also correlate with the appearance of saturable iron-binding sites on the surface of K562 cells with Kd approximately 0.4 microM. These results indicate that non-Tf iron transport activity and the expression of cell-surface iron-binding proteins can be controlled by environmental factors that promote megakaryocytic differentiation of K562 cells.

Biological Transport↗
PubMed1995

Cytosolic ADP-ribosylation factors are not required for endosome-endosome fusion but are necessary for GTP gamma S inhibition of fusion.

A specific role for ADP-ribosylation factors (ARFs) in in vitro endosome-endosome fusion has been proposed (Lenhard, J. M., Kahn, R. A., and Stahl, P. D. (1992) J. Biol. Chem. 267, 13047-13052). However, in vivo studies have failed to support a function for ARFs in the endocytic pathway, since an antagonist of ARF activities, brefeldin A, does not interfere with receptor internalization (Schonhorn, J. E., and Wessling-Resnick, M. (1994) Mol. Cell. Biochem. 135, 159-164). This controversy surrounding the exact function of ARF in endocytic vesicle traffic prompted us to critically re-examine the involvement of ARFs in cell-free endosome fusion. Cytosol depleted of ARF activity was capable of supporting in vitro endocytic vesicle fusion but failed to support inhibition of this reaction in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). Addition of purified ARF1 restored the ability of the ARF-depleted cytosol to inhibit endosome fusion when incubated with GTP gamma S. Both endocytic vesicle fusion and the GTP gamma S-mediated inhibition of vesicle fusion were unaffected by brefeldin A. Moreover, the ATP requirement and kinetics of cell-free fusion are not altered by brefeldin A or depletion of cytosolic ARFs. These results suggest that cytosolic ARFs are not necessary for endosomal vesicle fusion in vitro but are responsible for inhibition of fusion in the presence of GTP gamma S and cytosolic factors in a brefeldin A-resistant manner.

ADP-Ribosylation Factor 1↗
PubMed1995

Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants.

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

Amino Acid Sequence↗
PubMed1995

Mechanism of transferrin receptor down-regulation in K562 cells in response to protein kinase C activation.

Treatment with phorbol esters increases endocytosis of the transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and van Renswoude, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3005-3009). In this report, we demonstrate that this effect is reversible within early times of protein kinase C activation (< 2 h) but that prolonged exposure to phorbol esters results in a net loss of receptors. These effects are not due to the differentiation response of K562 cells to phorbol esters since bryostatin-1 also down-regulates the endocytosis of the transferrin receptor and shut downs receptor synthesis, but does not induce differentiation (Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P. (1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of receptor down-regulation which occur due to stimulation of receptor internalization from the cell surface. The fact that fluid-phase pinocytosis is also enhanced upon protein kinase C activation indicates that this effect is not specific for the transferrin receptor itself, but is a rather general cellular response to tumor-promoting phorbol esters. The fate of down-regulated transferrin receptors was followed in morphological and subcellular fractionation studies that demonstrate localization of this pool of receptors in early endocytic and recycling compartments. Our results exclude the possibility that transferrin receptor down-regulation results in trafficking of the receptor to lysosomal compartments for degradation. This idea is consistent with the observations that the time course of transferrin receptor degradation is not enhanced in stimulated K562 cells, while transferrin receptor synthesis is shut down. Our results rigorously demonstrate that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two stages: acute regulation of early steps in endocytosis that results in an immediate reduction of approximately 40% in cell surface number of receptors and a more chronic reduction in transferrin receptor synthesis upon prolonged exposure to phorbol esters (> 15 h).

Bryostatins↗
PubMed1995

Analysis of the stoichiometry of Rab protein prenylation.

Rab5 is a Ras-related GTP-binding protein that is post-translationally prenylated with the 20-carbon isoprenoid geranylgeranyl. We have developed a method to determine the stoichiometry of prenylation of Rab5, and Rab family members in general, based on the cell-free translation of these peptides in the presence or the absence of appropriate isoprenoids. Modification of cell-free synthesized Rab5 can be monitored by following the conversion of 35S-labeled peptide to a greater mobility isoform on urea-gradient sodium dodecyl sulfate-polyacrylamide gels. The mobility-shifted isoform also incorporates radiolabel in the presence of [3H]mevalonate or [3H]geranylgeranyl pyrophosphate, confirming post-translational modification with geranylgeranyl. A quantitative assessment of the conversion of mobility-shifted Rab5, promoted by prenylation, and the amount of incorporated radiolabel from [3H]geranylgeranyl pyrophosphate was achieved by excising gel slices containing radiolabeled isoforms and measuring the covalently associated radioactivity. Using this approach, we have established that 2 moles of geranylgeranyl is attached per mole of Rab5 peptide. A 2:1 molar ratio of geranylgeranyl:peptide is observed for both Rab5wt and a truncation mutant, Rab5(1-213), containing C-terminal motifs CCXX and XXCC, respectively. When Rab proteins ending in CXC are synthesized and processed in vitro, they also incorporate geranylgeranyl at a 2:1 stoichiometry, although extended times of incubation are required for full modification. Finally, a C-terminal Rab5 truncation mutant retaining only one cysteine also becomes modified, although only a minor fraction is fully processed. This method offers a novel, quantitative approach to investigate the stoichiometry of post-translational processing of cell-free synthesized peptides without the need to purify the native molecules.

Animals↗
PubMed1995

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.

Alkyl and Aryl Transferases↗
PubMed1995

Evidence for a low Km transporter for non-transferrin-bound iron in isolated rat hepatocytes.

Non-transferrin-bound iron (NTBI) plays an important role in the hepatocellular injury induced by iron overload. However, the mechanism responsible for NTBI uptake into hepatocytes remains poorly defined. The purpose of this study was to define the kinetics of NTBI uptake by isolated rat hepatocytes and to characterize the uptake process. NTBI uptake was time and temperature dependent, exhibited a Michaelis-Menten constant (Km) value of 1.25 microM and maximum uptake of 241 pmol.10(6) cells-1.min-1, and 55Fe was incorporated in part into intracellular ferritin. Uptake was Ca2+ dependent, exhibiting 15 and 80% of maximal uptake in the presence of 0.6 and 0.75 mM CaCl2, respectively. The putative NTBI transporter was highly specific; divalent (Zn2+, Mn2+, Cd2+, and Co2+) or trivalent (La3+) cations did not inhibit Fe3+ uptake. Reduction from Fe3+ to Fe2+ was not essential for uptake or the process occurred deep within the membrane bilayer, since the Fe2+ chelator ferrozine did not influence 55Fe uptake. These data provide evidence for a low Km plasma membrane transporter for NTBI, which should be functional at physiological serum concentrations and saturated in iron-overload diseases, such as hemochromatosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Extracellular ferrireductase activity of K562 cells is coupled to transferrin-independent iron transport.

The reduction of Fe3+ to Fe2+ has been established to play a critical role in the uptake of iron by many organisms. Recently, a mechanism of iron transport in the absence of transferrin (Tf) was described for the human K562 cell line and a role for ferrireductase activity was implicated in this process as well [Inman, R. S., & Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528]. The present report characterizes the extracellular reduction of ferricyanide to ferrocyanide catalyzed by K562 cells. The observation that membrane-impermeant ferricyanide competitively inhibits Tf-independent assimilation of iron from 55Fe-nitriloacetic acid indicates that this ferrireductase activity is indeed coupled to the transport mechanism. From a series of initial rate experiments, the kinetic parameters for cell surface ferrireductase activity, Vmax = 0.102 nmol min-1 (10(6) cells)-1 and Km = 6.13 microM, were determined. Neither the Vmax nor the Km of this reaction is modulated by changes in extra- or intracellular iron levels; thus, similar to Tf-independent transport activity in K562 cells, the ferrireductase activity is not regulated in response to iron levels. Transmembrane oxidoreductase activity is also reportedly involved in the control of cellular growth; however, the K562 cell ferrireductase is unresponsive to insulin and is not inhibited by the antitumor drugs adriamycin, actinomycin D, or cis-platin, observations that fail to support a role for this particular activity in cell regulation. Rather, the K562 cell ferrireductase appears to be tightly coupled to the mechanism of Tf-independent transport as demonstrated by its sensitivity to Cd2+, a specific inhibitor of non-Tf iron uptake by K562 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1994

Brefeldin A down-regulates the transferrin receptor in K562 cells.

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance ke, from 0.28 min-1 to 0.43 min-1, while BFA had little effect (Ke = 0.20 min-1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a 'slow-release', monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.

Brefeldin A↗
PubMed1993

Prenylation of Rab5 is dependent on guanine nucleotide binding.

Rab5 is a small molecular weight GTP-binding protein that functions in endocytic vesicle traffic. Like other Ras-related proteins, Rab5 is prenylated on C-terminal cysteine residues, although it lacks the typical C-terminal CAAX motif (where A is any aliphatic amino acid and X is any amino acid) to direct this post-translational modification. We have investigated structural requirements for the in vitro geranylgeranylation of Rab5. Rab5N133I, a point mutant that has impaired ability to bind GTP or GDP, undergoes modification to a limited extent and at a severely reduced rate when compared to cognate Rab5. A second point mutant, Rab5Q79L, can be processed to approximately the same extent as wild-type albeit at a reduced rate. Since the latter mutation results in defective GTPase activity, these combined observations indicate that guanine nucleotide binding plays an important role in the geranylgeranylation reaction and suggest that the GDP-bound form of Rab5 is the preferred conformation for interaction with Rab prenyltransferase. This idea is supported by the finding that non-hydrolyzable GTP analogs inhibit Rab5 prenylation, while in vitro processing of both H-ras and the gamma 2 subunit of regulatory G proteins is unaffected at concentrations of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) up to 400 microM. Moreover, a truncation mutant lacking the C-terminal cysteines, Rab5(1-211), serves as an inhibitor of Rab5wt geranylgeranylation when liganded with GDP but not GTP gamma S. Thus, the recognition of Rab5 as a substrate by Rab prenyltransferase involves structural elements exclusive of the C terminus and dependent upon the GDP-binding conformation of the protein.

Animals↗