IMMUNOCYTOLOGICAL LOCALIZATION OF AN EPITOPE-TAGGED PLASMA-MEMBRANE PROTON PUMP (H-ATPASE) IN PHLOEM COMPANION CELLS()

In higher plants, the plasma membrane proton pump (H+-ATPase) is encod ed by a surprisingly large multigene family whose members are expresse d in different tissues. Using an 18-amino acid epitope tag derived fro m the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AH A3 (Arabidopsis H+-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged A HA3 is restricted to the plasma membrane of phloem companion cells, wh ereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labe led tissue sections suggests that there is a high concentration of pro ton pumps in the plasma membrane of companion cells but a much lower c oncentration in the plasma membrane of sieve elements. Due to plasmode smata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pum p in companion cells can serve to power the uptake of sugar by proton- coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the met abolic energy that drives sugar uptake is consumed by AHA3 at the comp anion cell plasma membrane, These experiments with a genetically alter ed integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, o ur results demonstrate that, using genes encoding individual members o f a gene family, it is possible to label plasma membrane proteins immu nologically in specific, differentiated cell types of higher plants.