Proteolytic processing of CmPP36, a protein from the cytochrome b5 reductase family, is required for entry into the phloem translocation pathway

Cucurbita maxima (pumpkin) phloem sap contains a 31 kDa protein that cross-reacts with antibodies directed against the red clover necrotic mosaic virus movement protein (RCNMV MP). Microsequence data from phloem-purified 31 kDa protein were used to isolate a complementary DNA: the open reading frame encodes a 36 kDa protein belonging to the cytochrome b(5) reductase (Cb5R) family; the gene was termed CmPP36. Western analyses established that CmPP36, RCNMV MP and CmPP16 (Xoconostle-Cazares et al., 1999, Science 283, 94-98) are immunologically related, probably due to a common epitope, represented by the NADH(+)-binding domain of CmPP36. An N-terminal 5 kDa membrane-targeting domain is cleaved to produce the 31 kDa DeltaN-CmPP36 detected in the phloem sap. Microinjection experiments established that DeltaN-CmPP36, but not CmPP36, is able to interact with plasmodesmata to mediate its cell-to-cell transport. Thus, intercellular movement of CmPP36 requires proteolytic processing in the companion cell to produce a soluble, movement-competent, protein. In contrast to RCNMV and CmPP16, DeltaN-CmPP36 interacts with but does not mediate the trafficking of RNA. Northern and in site RT-PCR studies established that CmPP36 mRNA is present In all plant organs, being highly abundant within vascular tissues. In roots of hydroponically grown pumpkin plants, CmPP36 mRNA levels respond to changes in available iron in the culture solution. Finally, enzymatic assays established that both CmPP36 and DeltaN-CmPP36 could reduce Fe3+-citrate and Fe3+-EDTA in the presence of NADH(+). These findings are discussed in terms of the possible roles played by CmPP36 in phloem function.