Unusual subunits are directly involved in binding substrates for natural rubber biosynthesis in multiple plant species

Rubber particles from rubber-producing plant species have many different species-specific proteins bound to their external monolayer biomembranes. To date, identification of those proteins directly involved in enzymatic catalysis of rubber polymerization has not been fully accomplished using solubilization, purification or reconstitution approaches. In an alternative approach, we use several tritiated photoaffinity-labeled benzophenone analogs of the allylic pyrophosphate substrates, required by rubber transferase (RT-ase) to initiate the synthesis of new rubber molecules, to identify the proteins involved in catalysis. Enzymatically-active rubber particles were purified from three phylogenetically-distant rubber producing species, Parthenium argentatum Gray, Hevea brasiliensis Muell. Arg, and Ficus elastica Roxb., each representing a different Superorder of the Dicotyledonae. Geranyl pyrophosphate with the benzophenone in the para position (Bz-GPP(p)) was the most active initiator of rubber biosynthesis in all three species. When rubber particles were exposed to ultra-violet radiation, 95% of RT-ase activity was eliminated in the presence of 50 mu M Bz-GPP(p), compared to only 50% of activity in the absence of this analog. H-3-Bz-GPP(p) then was used to label and identify the proteins involved in substrate binding and these proteins were characterized electrophoretically. In all three species, three distinct proteins were labeled, one very large protein and two very small proteins, as follows: P. argentatum 287,000, 3,990, and 1,790 Da; H. brasiliensis 241,000, 3,650 and 1,600 Da; F. elastica 360,000, 3,900 and 1,800 Da. The isoelectric points of the P. argentatum proteins were 7.6 for the 287,000 Da, 10.4 for the 3,990 Da and 3.5 for the 1,790 Da proteins, and of the F. elastica proteins were 7.7 for the 360,000 Da, 6,0 for the 3,900 Da, and 11.0 for the 1,800 Da proteins. H. brasiliensis protein pI values were not determined. Additional analysis indicated that the three proteins are components of a membrane-bound complex and that the ratio of each small protein to the large one is 3:1, and the large protein exists as a dimer. Also, the large proteins are membrane bound whereas both small proteins are strongly associated with the large proteins, rather than to the rubber particle proteolipid membrane.