Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis.
Year: 1994
Type: article
Abstract: Rab5 is a Ras-like small GTPase that regulates early events of endocytosis.Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and RabS:N1331) are dominant inhibitors of endocytosis.In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants.Second-site mutations were introduced into Rab5:S34N and Rab5:N1331, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector.Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase).The effects of the Rab5 mutants on endosome fusion in vitro were also examined.Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N1331.The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain.Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:NlSBI.The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras.This mutation greatly decreased the isoprenylation of RabZN133I and its inhibitory activity on endocytosis.It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors.Some of these interactions are disrupted by Rab5:S34N and Rab5:N1331.By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor.This interaction requires the effector domain Phe" residue and C-terminal isoprenylation of Rab5.The Rab family of Ras-related small GTPases regulates intracellular membrane trafficking (for a recent review, see Ref. 1).These proteins are specifically localized to the cytoplasmic surface of each intracellular compartment, such as endoplasmic reticulum (Rabl and Rab2) (2, 3), cis-Golgi (Rabl and Rab2) (2, 31, medial-Golgi (Rab6) (4), trans-Golgi (Rab9) (5), early endosome (Rab4 and Rab5) (2, 6), late endosome (Rab7 and Rab9) (2,5), and plasma membrane (Rab5) (2).While most
Source: Journal of Biological Chemistry
Authors: G. Li, M.A. Barbieri, M.I. Colombo, P.D. Stahl
Institution: Washington University in St. Louis
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1563901359