# Biosynthesis of Natural Rubber: Current State and Perspectives
## 2 Biosynthesis of Natural Rubber and the Involved Genes/Proteins
### Genes/Proteins Involved in Natural Rubber Biosynthesis
#### 2.3.3. CPT-Like/CPT-Binding Protein  
CPT-Like/CPT-binding proteins (CPTL/CPTBP) are the most recently isolated proteins that are involved in rubber biosynthesis which attracts great interest. They are homologs of the human Nogo-B receptor (NgBR), and localized mainly on the ER in non-rubber-producing eukaryotes and on both ER and rubber particles in rubber-producing plants [44; 48; 79; 80; 81]. The NgBR homologs usually contain one or more transmembrane domains in the N-terminal region and a low-similarity CPT-like domain in the C-terminal half [11; 38]. Although they do not have catalytic residues conserved among CPTs, the RxG motif in their C-terminal was demonstrated to be critical for prenyltransferase activity [82]. AtLEW1 (LEAF WILTING1) was reported to be required for the dolichol biosynthesis and mediates plant responses to ER stress, drought, and dark-induced senescence in _Arabidopsis_[83]. Characterization of lettuce _cis_-prenyltransferase-like 2 (LsCPTL2) suggested that LsCPTL2 is a scaffold protein tethering LsCPT3 on ER and is necessary for natural rubber biosynthesis in lettuce [48]. However, yeast-expressed LsCPTL2 and LsCPT3 alone could not synthesize high-molecular-weight natural rubber in vitro without rubber particles, indicating other components may be required for the rubber biosynthetic machinery [48]. It was also proved that the tomato SICPT3 and its partner CPT-binding protein (SICPTBP) interact in vivo and both of them are strictly required for dolichol synthase activity [81]. The _T. brevicorniculatum_ rubber transferase activator (TbRTA) was demonstrated to be an essential component of the rubber transferase complex which interacts with TbCPTs on the surface of rubber particles [43]. Its knockdown mutants showed decreased abundance of rubber particles and eliminated rubber biosynthesis [43].  
[PERSON] et al. made progress in identifying the components of the rubber transferase complex by reconstituting rubber biosynthetic machinery on detergent-washed _H. brasiliensis_ rubber particles (WRPs) in vitro [38]. HRT1-REF BRIDGING PROTEIN (HRBP), which belongs to the CPTL/CPTBP clade (Figure 3), was demonstrated to play important roles in introducing recombinant CPTs to WRPs. It interacts with HRT1/2 and REF, and may act as a bridge between HRT1 and REF to form a complex [38; 44]. Co-expression of HRBP and REF decreased the amount of HRT1 which was not incorporated on WRPs, indicating they promote the incorporation of HRT1 into WRPs [38]. Introducing HRT1 onto WRPs distinctly increased the rubber transferase activity, whereas HRBP or REF alone did not activate the background activity of WRPs [38]. Co-expression of HRBP or both HRBP and REF could enhance the HRT1-derived rubber transferase activity, but had no effect on product chain length regulation [38]. Co-expression results of these proteins in _Nicotiana benthamiana_ leaves showed that the HRT1/HRBP/REF complex primarily was localized on ER and ER-emerged-specific particles which supported the idea that rubber particles may originate from ER membrane [38]. Therefore, from the interaction network, rubber transferase activity assay and subcellular localization analyses, it is suggested that the natural rubber biosynthetic machinery consists of CPT, HRBP and REF [38; 44] (Figure 4). As HRBP and REF have no catalytic domain or activity, they may function as structural proteins stabilizing the rubber biosynthetic machinery on rubber particles.