The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

Abstract

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

“…Although the isoprenylated C-terminal motif is required for membrane targeting (66,67), functions of the extreme N-terminal region are still unknown. Given that deletion of the first 14 -22 amino acid residues at the N-terminal domain alone is enough to disrupt Rab5a functionalities (42), the N-terminal region is not only an important element for Rab5a function; it might also act as the domain that specifies isoform functionality. Taking it a step further, we have shown that when phosphorylation of Rab5a on the N-terminal Thr-7 site is disrupted, T-cell migration through ICAM-1-coated membranes toward SDF-1␣ is significantly repressed.…”

Co‐operative regulation of endocytosis by three RAB5 isoforms

“…The assay was performed essentially as described [20,31]. Briefly BHK cells were allowed to internalize avidin or biotinylated horseradish peroxidase (bHRP).…”

“…The C-terminal domain functions in targeting the protein to the early endosomes but it is not sufficient to confer Rab5a function [34,37]. The Nterminus and the regions predicted to undergo nucleotidedependent conformational changes (the effector domain or switchl, ~2/L5 or switch2 and ct3/L7) are additionally required for the regulatory role of Rab5a in endocytic transport [31,37]. Ca-Rab5a…”

Influence of Mg2+ on the Structure and Function of Rab5

“…The synthetic protein binds endogenous GDP (40); limited digestion of this GDP-bound form with trypsin produces a single 35 S-labeled fragment of 14 kDa. Addition of 30 mM EDTA to chelate Mg 2ϩ essential for guanine nucleotide binding markedly reduces the amount of the latter tryptic peptide.…”

“…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)(34)(35)(36)(37)(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).…”