Rab7 and Rab9 are recruited onto late endosomes by biochemically distinguishable processes.
Rab GTPases are localized to the surfaces of distinct membrane-bound organelles and function in transport vesicle docking and/or fusion. Prenylated Rab9, bound to GDP dissociation inhibitor-alpha, can be recruited selectively onto a membrane fraction enriched in late endosomes; this process is accompanied by nucleotide exchange. We used this system to address whether each Rab uses a distinct machinery to associate with its cognate organelle. Purified, prenylated Rab1B, Rab7, and Rab9 proteins were each reconstituted as stoichiometric complexes with purified GDP dissociation inhibitor-alpha, and their recruitment onto endosome- or ER-enriched membrane fractions was quantified. The two late endosomal proteins, Rab9 and Rab7, were each recruited onto endosome membranes with approximate apparent Km values of 9 and 22 nM, respectively. However, while control Rab9.GDP dissociation inhibitor-alpha complexes inhibited the initial rate of myc-tagged Rab9 recruitment with an apparent Ki of approximately 9 nM, Rab7 complexes inhibited this process much less effectively (apparent Ki approximately 112 nM). Similarly, complexes of the endoplasmic reticulum-localized Rab1B protein were even less potent than Rab7 complexes (apparent Ki approximately 405 nm). Rab9 complexes inhibited Rab7 recruitment with the same low efficacy as Rab7 complexes inhibited Rab9 recruitment. These experiments distinguish, biochemically, the recruitment of different Rab proteins onto a single class of organelle. Since Rab7 and Rab9 are both localized at least in large part, to late endosomes, this suggests that a single organelle may bear multiple Rab recruitment machines.
Soldati
T
T
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
Rancaño
C
C
Geissler
H
H
Pfeffer
S R
SR
D000818:Animals; D001667:Binding, Competitive; D015415:Biomarkers; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004285:Dogs; D011992:Endosomes / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D007700:Kinetics; D011485:Protein Binding; D017368:Protein Prenylation; D013347:Subcellular Fractions / Q000378:metabolism; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D015415:Biomarkers; D003432:Cross-Linking Reagents; D020730:Guanine Nucleotide Dissociation Inhibitors; D000090802:rab7 GTP-Binding Proteins; C000716466:rab7 GTP-binding proteins, rat; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C061569:RAB1B protein, rat; C489802:Rab9a protein, rat; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins