Abstract
Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP- but not GDP-saturated Rab3 was localized to a short alpha-helical sequence near the Rim1 N terminus (amino acids 19-55). Rab3 isoforms A, C, and D were bound with similar affinities (K(d) = 1-2 microm). Low affinity binding of Rab6A-GTP was also observed (K(d) = 16 microm), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal alpha-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K(d) on the order of magnitude of 10-100 microm) to this region of rabphilin-3 alone (amino acids 40-88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K(d) = 10-20 nm). In contrast, an N-terminal alpha-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to downstream Rim1 sequences, between amino acids 56 and 387, of syntaxin and of Rab3. The latter, however, was inhibited rather than stimulated by GTP.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ310531 and AJ310532
Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics
Recombinant mouse Rim1 sequences encompassing various parts of the N-terminal 387 amino acids (Fig. 1) were fused to glutathione S-transferase (GST) and expressed in bacteria, and their interaction with bacterially expressed, His6-tagged recombinant Rab3A was studied by Surface Plasmon Resonance (SPR) (Fig. 2, Table I)
Summary
Recombinant Proteins—Full-length coding sequences of Rab proteins, from codon 2 to the stop codon, were amplified by reverse transcription-polymerase chain reaction from mouse brain RNA and inserted into the SmaI site of the His tag vector pQE32 (Qiagen). Recombinant Rab proteins were expressed and purified under nondenaturing conditions following Qiagen protocols, with the addition of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 2 g/ml pepstatin A, 2 g/ml leupeptin) to the lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole). GST fusion proteins with Rim, aczonin, and rabphilin-3 partial sequences were immobilized at surface densities of 500 – 800 RUs on an anti-GST antibody-coated surface, and the interaction with Rab proteins in rising concentration order was analyzed at 20 °C and at a flow rate of 30 l/min in association and dissociation, again in buffer A with 3 mM MgCl2 and 0.5 mM GTP␥S or 1 mM GDP. Additional zinc finger-protecting measures in control experiments (inclusion of 10 M ZnCl2 and 5 mM DTT in the preincubation and dilution buffers) did not enhance the Rab3A binding of Rim zinc finger constructs.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
