Proteomic and targeted lipidomic analyses of fluid and rigid rubber particle membrane domains in guayule

Authors: BLAKESLEE, JOSHUA - The Ohio State University; HAN, EUN-HYANG - The Ohio State University; LIN, YUN - The Ohio State University; LIN, JINSHAN - The Ohio State University; NATH, SEEMA - The Ohio State University; ZHANG, LIWEN - The Ohio State University; LI, ZHENYU - The Ohio State University; Cornish, Katrina

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2024
Publication Date: 10/24/2024
Citation: Blakeslee, J.J., Han, E., Lin, Y., Lin, J., Nath, S., Zhang, L., Li, Z., Cornish, K. 2024. Proteomic and targeted lipidomic analyses of fluid and rigid rubber particle membrane domains in guayule. Plants. 13(21):2970. https://doi.org/10.3390/plants13212970.
DOI: https://doi.org/10.3390/plants13212970

Interpretive Summary: The identification of the components of the enzyme complex that makes natural rubber (RT-ase) is very difficult because activity requires that the complex is embedded in the rubber particle membrane. We hypothesized that if detergent resistant membrane rafts were present in rubber particles that these would be the likely place to find RT-ase. To investigate this hypothesis, the lipid and protein profiles of RP membranes and DRMs were characterized and interpreted and several new proteins have been identified which may be essential to rubber biosynthesis.

Technical Abstract: Rubber (cis-1,4-polyisoprene) is produced in cytosolic unilamellar vesicles called rubber particles (RPs), and the protein complex responsible for this synthesis, the rubber transferase (RTase), is embedded in, or tethered to, the membranes of these RPs. Solubilized enzyme activity is very difficult to achieve because the polymerization of highly hydrophilic substrates into hydrophobic polymers requires a polar/non-polar interface and a hydrophobic compartment. Using guayule (Parthenium argentatum) as a model rubber-producing species, we optimized methods to isolate RP unilamellear membranes and then a subset of membrane microdomains (detergent-resistant membranes) likely to contain protein complexes such as RTase. The phospholipid and sterol composition of these membranes and microdomains were analyzed using thin-layer chromatography (TLC) and liquid chromatography tandem mass spectroscopy (LC-MS/MS). Our data indicate that RP membranes consist predominantly of phosphatidic acid-containing membrane microdomains (DRMs or “lipid rafts”). Proteomic analyses of guayule RP membranes and membrane microdomains identified 80 putative membrane proteins covering 30 functional categories. From this population, we have tentatively identified several proteins in multiple functional domains associated with membrane microdomains which may be critical to RTase function. Definition of the mechanisms underlying rubber synthesis will provide targets for both metabolic engineering and breeding strategies designed to increase natural rubber production in latex-producing species.