Entry created on 1 July 2019 (Revision 1.0) Annotator: Nikoletta Murvai
This entry is part of a multi-component system encompassing the following entries: Q94ET8 P00873
Basic protein information
Accession Q94ET8
Common name EPYC1
Gene LCI5
Organism Chlamydomonas reinhardtii
Uniprot name LCI5
Basic LLPS information
Organelle pyrenoid
Type of experimental evidence
Joined entry Q94ET8 P00873
Protein region(s) mediating LLPS
52
-
291
Four almost identical ~60 amino-acid tandem repeats in EPYC1
Based on the experimental results of the following publication: 27166422
Molecular features viewer
PDB structures
Extended LLPS information
Functional description
The pyrenoid is a carbon-fixing organelle in algae that undergoes LLPS owing to multivalent interactions between Rubisco and Essential Pyrenoid Component 1 (EPYC1). Rubisco and the linker protein EPYC1, are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Rubisco has eight binding sites for EPYC1, while EPYC1 has four binding sites for Rubisco. Modeling suggests that such systems will exhibit a magic number effect where certain numbers of particles form an unusually stable state. The magic number effect manifests when the valency of one partner is an integral multiple of the valency of the second and the binding sites of the two partners can be saturated. This magic number effect could impact the phase diagram in many biological contexts and is predicted to give rise to unexpectedly sharp phase transition (PMID:30951647) If each repeat of EPYC1 binds Rubisco, then EPYC1 could link multiple Rubisco holoenzymes together to form the pyrenoid matrix. Multiple Rubisco binding sites on EPYC1 could arrange Rubisco into the hexagonal closely packed or cubic closely packed arrangement observed in recent cryoelectron tomography studies of the Chlamydomonas pyrenoid. EPYC1 and Rubisco could interact in one of two fundamental ways: (i) EPYC1 and Rubisco could form a codependent network, or (ii) EPYC1 could form a scaffold onto which Rubisco binds. Importantly, the 60-aa repeat length of EPYC1 is sufficient to span the observed 2- to 4.5-nm gap between Rubisco holoenzymes in the pyrenoid, and a stretched-out repeat could potentially span the observed 15-nm Rubisco center-to-center distance (PMID:27166422).
Literature supporting the LLPS: 27166422, 28938114, 30498228, 30675061, 31001862
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor
Binding partners (at biological protein concentrations)
1) Rubisco (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:28938114) electrostatic (cation-anion) interaction (PMID:30498228)
Determinants of phase separation and droplet properties
1) protein concentration of EPYC1 in the presence of crowding agents 2) stochiometry of the components 3) salt concentration
Membrane cluster No
Partner-dependent No
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
Mixing pure C. reinhardtii Rubisco and EPYC1 led to immediate formation of a turbid solution that cleared over time in vitro. The turbidity was caused by the formation of spherical droplets (morphology) from the bulk solution that could be labeled by including a fluorescent EPYC1-GFP fusion protein in the reaction. The observed clearance of the solution was caused by fusion of the droplets into a large homogeneous droplet (coalescence), supporting their liquid nature. Demixed droplets could be harvested by centrifugation, and SDS-polyacrylamide gel electrophoresis analysis confirmed that both EPYC1 and Rubisco had entered the droplets (co-localization) (PMID:30498228). To confirm the pyrenoid localization of EPYC1, the authors coexpressed fluorescently tagged EPYC1 and RBCS. Venus-tagged EPYC1 showed clear colocalization with mCherry-tagged RBCS in the pyrenoid in vivo (PMID:27166422). Cryo-ET (imaging assay evidence) measurements revealed that the pyrenoid matrix is not crystalline, but exhibits liquid-like local order (morphology) and FRAP experiments revealed that the pyrenoid matrix mixes internally. The Chlamydomonas pyrenoid matrix also appears to undergo such a phase transition during division: a portion of the RBCS1-Venus and EPYC1-Venus signals rapidly dispersed from the pyrenoid matrix into the stroma investigated by fluorescent microscopy (PMID:28938114).
Experimental observations supporting the liquid material state of the condensate
dynamic movement/reorganization of molecules within the droplet (PMID:28938114) dynamic exchange of molecules with surrounding solvent (PMID:30498228) morphological traits (PMID:30498228)