Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles

Abstract

Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).