A PHLOEM-SPECIFIC SUCROSE-H-MAJOR L SUPPORTS THE MODEL OF APOPLASTIC PHLOEM LOADING( SYMPORTER FROM PLANTAGO)

In this paper the cloning of a full-length cDNA clone encoding the PmS UC2 sucrose-H+ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regio ns of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)() RNA isolated from vascular bundles and used for the subsequent cloni ng of cDNAs. The respective mRNA is specifically expressed in the vasc ular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembran e helices and is highly homologous to other plant sucrose transporters . Substrate specificity and energy dependence of the transporter encod ed by this cDNA were determined by expression in baker's yeast Sacchar omyces cerevisiae. The PmSUC2 protein catalyses the transport of sucro se into transgenic yeast cells. Invertase null mutants of yeast expres sing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to p roteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H+ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The pr esented data strongly support the theory of phloem loading from the ap oplastic space by a sucrose-H+ symporter.