Evidence for Sequential Action of Rab 5 and Rab 7 GTP ases in Prevacuolar Organelle Partitioning

GTP ases of the Rab 5 and Rab 7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant‐specific Rab 5 reside at the newly discovered ‘late prevacuolar compartment’ ( LPVC ) while Rab 7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild‐type Rab 5 GTP ases but not Rab 7 lead to dose‐dependent inhibition of biosynthetic vacuolar transport. In the case of Ara 6, this included aberrant co‐localization with markers for earlier post‐ G olgi compartments including the trans ‐ G olgi network. However, nucleotide‐free mutants of all three GTP ases ( Rha 1, Ara 6 and Rab 7) cause stronger dose‐dependent inhibition of vacuolar sorting. In addition, nucleotide‐free Rha 1 led to a later maturation defect and co‐localization of markers for the prevacuolar compartment ( PVC ) and the LPVC . The corresponding Rab 7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab 5 family members is underlined by the fact that mutant Rha 1 expression can be suppressed by increasing wild‐type Rha 1 levels while mutant Ara 6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab 5 and Rab 7 GTP ases is presented in the light of the current observations.