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.

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal 59Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.

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 (Rab523-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab523-215) 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-yS. 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.

Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments (1, 2) and believed to regulate specific steps of intracellular membrane trafficking (3-6). The functional cycle of Rab proteins involves the delivery of the GDP-bound forms to the target membrane by a GDP dissociation inhibitor (GDI) 1 (7-9), the exchange of GDP for GTP at membrane surface catalyzed by a guanine nucleotide exchange factor (GEF) (8, 9) and the retrieval of the GDP-bound forms from the membrane by GDI after GTP hydrolysis and membrane fusion (7). Localized on plasma membrane, clathrin-coated vesicles, and early endosomes (2), Rab5 has been shown to play an important role in early events of endocytosis (4, 5), although the exact mechanism of its function remains to be determined.It is known that Mg 2ϩ is essential for GTPase function and structure. Crystallographic studies of several GTP-binding proteins reveal a single Mg 2ϩ in the guanine nucleotide binding pocket, coordinating between the protein and guanine nucleotide in both GDP-and GTP analog-bound conformations (10 -15). Effects of Mg 2ϩ on guanine nucleotide binding, GTPase activity, and the structural integrity of GTP-binding proteins have been widely documented (16 -32). A key observation is that Mg 2ϩ inhibits GDP release from Ras-like GTP-binding proteins and therefore prevents binding of . However, the exact mechanism for this inhibitory effect and its physiological significance remains unknown.Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins also have been noted in studies of guanine nucleotide exchange (33-38). Myristoylation at the N terminus of ARF enhances its rate of GDP release (27), and N-terminal truncation of ARF results in loss of function by reducing its affinity for GDP and permitting GDP/GTP exchange in the absence of phospholipids (33). Moreover, deletion of the N terminus enables isolation of ARF in a nucleotide-free form (38). Finally, deletion of the N-terminal domain of Rab5 results in a loss of function (34 -37) and interferes with the protein's post-translational processing (37). These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins.We have investigated mechanisms through which Mg 2ϩ and the N-terminal domain of Rab5 participate in its regulation of GDP release. . While the structure and function of Rab5 , a C-terminal truncation mutant, is influenced by Mg 2ϩ in the same fashion as Rab5WT , an N-and C-terminal truncation mutant, Rab5 , is resistant to the cation's effects. Thus, inhibition of GDP release by Mg 2ϩ appears to be exerted via chemical constraints due to the cation's coordination between GDP and Rab5, as well as conformational restraints involving the protein's N-terminal domain that are induced by Mg 2ϩ coordination with Ser 34 of Rab5. Based on the correl...

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