RAB7 AND RAB9 ARE RECRUITED ONTO LATE ENDOSOMES BY BIOCHEMICALLY DISTINGUISHABLE PROCESSES

Rab GTPases are localized to the surfaces of distinct membrane-bound o rganelles and function in transport vesicle docking and/or fusion. Pre nylated Rab9, bound to GDP dissociation inhibitor-alpha, can be recrui ted selectively onto a membrane fraction enriched in late endosomes; t his process is accompanied by nucleotide exchange. We used this system to address whether each Rab uses a distinct machinery to associate wi th its cognate organelle. Purified, prenylated Rab1B, Rab7, and Rab9 p roteins were each reconstituted as stoichiometric complexes with purif ied GDP dissociation inhibitor-alpha and their recruitment onto endoso me- or ER-enriched membrane fractions was quantified. The two late end osomal proteins, Rab9 and Rab7, were each recruited onto endosome memb ranes with approximate apparent K-m values of 9 and 22 nM, respectivel y. However, while control Rab9 . GDP dissociation inhibitor-alpha comp lexes inhibited the initial rate of myc-tagged Rab9 recruitment with a n apparent K-i of approximate to 9 nM, Rab7 complexes inhibited this p rocess much less effectively (apparent K-i approximate to 112 nM). Sim ilarly, complexes of the endoplasmic reticulum-localized Rab1B protein were even less potent than Rab7 complexes (apparent K-i approximate t o 405 nM). Rab9 complexes inhibited Rab7 recruitment with the same low efficacy as Rab7 complexes inhibited Rab9 recruitment. These experime nts distinguish, biochemically, the recruitment of different Rab prote ins onto a single class of organelle. Since Rab7 and Rab9 are both loc alized at least in large part, to late endosomes, this suggests that a single organelle may bear multiple Rab recruitment machines.