Phosphate-binding loop and Rab GTPase function: mutations at Ser(29) and Ala(30) of Rab5 lead to loss-of-function as well as gain-of-function phenotype

Ras-like GTPases contain a structurally conserved GTP-binding domain. An im portant element of the GTP-binding domain is the phosphate-binding loop, wh ich contains two Gly residues (Gly(12) and Gly(13)) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that th ey are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser(29) and Ala(30). The p resent study builds On earlier biochemical characterizations of the Rab5 mu tants containing substitutions at Ala(30) and provides a comprehensive anal ysis of the structure-function relationship of the Rab5 phosphate-binding l oop. We have generated 19 new mutants containing amino acid substitutions a t Ser29 and determined whether these Ser(29) mutants, as well as the Ala(30 ) mutants, remain able to stimulate the endocytosis elf horseradish peroxid ase in baby hamster kidney cells. A total of 11 mutants lose the activity o f stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachme nt from culture dishes. They also induce super-large early endosomes such a s the constitutively activated Rab5:Q79L mutant. Biochemical results sugges t that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes fun ctional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-fun ction phenotype, indicating a structure-function relationship distinct from that of Ras.