Entry created on 1 July 2019 (Revision 1.0) Annotator: Rawan Abukhairan
This entry is part of a multi-component system encompassing the following entries: Q9JIR4 Q9JIR1
Basic protein information
Accession Q9JIR4
Common name RIM1
Gene RIMS1
Organism Rattus norvegicus
Uniprot name Regulating synaptic membrane exocytosis protein 1
Basic LLPS information
Organelle cytoplasmic protein granule
Type of experimental evidence
Joined entry Q9JIR4 Q9JIR1
Protein region(s) mediating LLPS
505
-
1206
IDR with P-rich motifs called PRM1, PRM2 and PRM
Based on the experimental results of the following publication: 30661983
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
RIM1 and RIM-BP2 are two major scaffold proteins in synaptic transmissions located in the active zone. The two proteins together can undergo LLPS in vitro and the formed condensates cluster Ca²⁺ channels in solution and on membrane surface, this may be a key-finding to understand how presynaptic active zones form and function to regulate neurotransmitter release. Multivalent interactions between RIM1 and RIM-BP2 and their intrinsically disordered properties lead to the formation of self-organized, highly condensed and dynamic assemblies that are reminiscent of dense projection-like structures through liquid-liquid phase separation (LLPS) in vitro. In vitro study showed that RIM1 alone at high concentrations could undergo LLPS, and this LLPS is sensitive to the salt concentration in the assay buffer. RIM1 is the key determinant of the formation of RIM1/RIM-BP2 condensates. RIM1/RIM-BP2 LLPS is driven by the binding of the proline rich motifs (PRMs) within RIM1 sequence to the three SH3 domains of RIM-BP2. The formed condensed phase may act as a platform to recruit other scaffold proteins and signaling proteins, including ELKS, liprins, Munc13, and Rab3/27 in presynaptic termini. These condensates also cluster Ca²⁺ channels in solution and on membrane surface. N-type and P/Q-type Voltage-gated Ca²⁺ channels (VGCCs) directly bind to RIM1 and RIM-BP2 via their cytoplasmic tails, such binding significantly promotes LLPS of RIM1 and RIM-BP2 as well as enriches VGCCs to the condensed liquid phase. PRM or PBM of VGCCs are responsible for such behaviour as they drive multivalent interactions. Proteins concentration appeared to affect the clustering patterns of RIM, RIM- BP, and VGCC on supported lipid bilayer but does not affect their patterns in solution. As a conclusion, the presynaptic active zone is formed through LLPS where RIMs and RIM-BPs are considered as plausible organizers of active zones, and their condensates can cluster VGCCs into nano- or microdomains and position them with Ca²⁺ sensors on docked vesicles for efficient and precise synaptic transmissions (PMID:30661983).
Literature supporting the LLPS: 30661983, 30849390
Functional class of membraneless organelle: regulator of spatial patterns; activation/nucleation/signal amplification/bioreactor
Binding partners (at biological protein concentrations)
1) RIM-BP (strictly required for LLPS) 2) N-type and P/Q-type Voltage-gated Ca²⁺ channels (promotes LLPS)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required
Molecular interaction types contributing to LLPS
multivalent domain-motif interactions (PMID:30661983)
Determinants of phase separation and droplet properties
1) protein concentration of Rimbp 2) salt concentration (for RIM self-LLPS)
Membrane cluster No
Partner-dependent Yes
RNA-dependent No
PTM required No
Domain-motif interactions Yes
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
Purified RIM1α-PAS (PDZ-C2A-PRM) and the SH3 domains of RIM-BP2 (i.e., by deleting the three FN3 [fibronectin type III] domains due to their hitherto unknown functions) were used to study their physical interaction in vitro. Using sedimentation-based assay, it was found that mixing these two proteins at different molar ratios led to LLPS of both proteins. Fluorescent tagging of purified RIM1α- PAS and RBP2-(SH3)3 was done and when they were mixed, differential interference contrast (DIC) microscopy and fluorescence images have shown protein co-localization and enrichment in condensed droplets in vitro (protein localization). Droplet fusion events and fluorescence recovery after photobleaching (FRAP) confirmed the liquid state of the condensed droplets (morphology). Sedimentation-based assays showed that truncation of different PRM regions (PRM1: D502–510; PRM2: D873–876; PRM: D1,086–1,089) weakened or even abolished LLPS of RIM1α-PAS when mixed with RBP2-(SH3)3 (particle size and count), indicating that all three PRMs contribute to LLPS. An ITC-based assay showed that the RIM1α PRM2 and a stretch of disordered sequences following PRM2 could indeed bind to RBP2-(SH3)3. The model of RIM1α-PAS and RBP2-(SH3)3 LLPS was tested via the multivalent interaction between the two proteins: LLPS experiments were performed by titrating increasing amounts of RBP2-(SH3)3 to a fixed concentration of RIM1α-PAS (change in protein concentration). This titration experiment indicated that a high concentration of RBP2-(SH3)3 titrated away the large RIM1α/RBP2-(SH3)3 species and thus dispersed the formed RIM1α/RBP2-(SH3)3 droplets, an observation fitting the multivalent protein-protein-interaction-mediated LLPS model. ITC measurment showed that the segment encompassing the two proline-rich regions (aa 183–480 not found in RIMa-PAS), but not the zinc-finger domain, indeed binds to RBP2-(SH3)3, albeit with a relatively weak affinity. Sedimentation-based assay showed that when RIM1α-FL and RBP2-(SH3)3 were mixed at a 1:1 molar ratio, the mixture underwent LLPS at a concentration as low as 2.5 mM, indicating that the N-terminal proline-rich sequences indeed promote LLPS of RIM1α with RBP2-(SH3)3. DIC and fluorescence microscopy have shown RIM1α-FL and RBP2-(SH3)3 protein co-localization and enrichment in condensed droplets. FRAP experiment indicated the existence of a less mobile fraction of RIM1α-FL in the condensed droplet. Imaging-based assays and ITC experiments showed NCav-CT (cytoplasmic tail of the N-type VGCC alpha1 subunit) could be enriched (co-localization) and in return promote LLPS of RIM1α-FL and RBP2-(SH3)3.
Experimental observations supporting the liquid material state of the condensate
dynamic movement/reorganization of molecules within the droplet (PMID:30661983) morphological traits (PMID:30661983)