Functional determinants of ras interference 1 mutants required for their inhbitory activity on endocytosis.
In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay. In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.
Galvis
Adriana
A
Department of Biological Sciences, Florida International University 11220 SW 8(th) Street, Miami, FL 33199, USA.
Giambini
Hugo
H
Villasana
Zoilmar
Z
Barbieri
M Alejandro
MA
D000818:Animals; D002478:Cells, Cultured; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000235:genetics*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008561:Membrane Fusion / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D018411:Spodoptera; D013329:Structure-Activity Relationship; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; C092117:RIN1 protein, human; D004815:Epidermal Growth Factor; D020696:rab5 GTP-Binding Proteins