Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis.
The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.
Dennis
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MS
Department of Medicinal and Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
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D000485:Allergens; D052179:Antigens, Plant; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D004122:Dimethylallyltranstransferase / Q000378:metabolism*; D045782:Hemiterpenes; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D007840:Latex / Q000032:analysis / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010940:Plant Proteins; D010944:Plants / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D012408:Rubber / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism*; D014197:Trees; D014357:Trypsin / Q000494:pharmacology
D000485:Allergens; D052179:Antigens, Plant; D003902:Detergents; D045782:Hemiterpenes; D007074:Immunoglobulin G; D007840:Latex; D009943:Organophosphorus Compounds; D010940:Plant Proteins; D011506:Proteins; C061477:REF protein, Hevea brasiliensis; C004809:isopentenyl pyrophosphate; D012408:Rubber; D014166:Transferases; D004122:Dimethylallyltranstransferase; D014357:Trypsin