Mechanism of association and reciprocal activation of two GTPases.

Shu-Ou Shan,Robert M Stroud,Peter Walter,Fred Hughson Fred Hughson

doi:10.1371/journal.pbio.0020320

Abstract

The signal recognition particle (SRP) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane. During this process, two GTPases, one in SRP and one in the SRP receptor (named Ffh and FtsY in bacteria, respectively), form a complex in which both proteins reciprocally activate the GTPase reaction of one another. Here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the Ffh-FtsY interaction. Mutations of a large number of residues at the interface impair complex formation, supporting the importance of an extensive interaction surface. Surprisingly, even after a stable complex is formed, single mutations in FtsY can block the activation of GTP hydrolysis in both active sites. Thus, activation requires conformational changes across the interface that coordinate the positioning of catalytic residues in both GTPase sites. A distinct class of mutants exhibits half-site reactivity and thus allows us to further uncouple the activation of individual GTPases. Our dissection of the activation process suggests discrete conformational stages during formation of the active SRP•SRP receptor complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.

Highlights

GTPases comprise a superfamily of proteins that provide molecular switches to regulate many cellular processes, including translation, signal transduction, cytoskeletal organization, vesicle transport, nuclear transport, and spindle assembly (Gilman 1987; Bourne et al 1991)
Dissection of the mutational effect on individual steps allows us to divide the deleterious mutants into distinct classes: Class I mutants primarily affect complex formation, Class II mutants primarily affect the reciprocal GTPase activation, Class III mutants are defective in both steps, and Class IV or half-site mutants block the activation of only one GTPase site in the complex (Table 1)
The protein concentration dependence of this reaction further indicates that the defects in these mutants can be functionally distinguished, allowing us to group them into different classes

Summary

Introduction

GTPases comprise a superfamily of proteins that provide molecular switches to regulate many cellular processes, including translation, signal transduction, cytoskeletal organization, vesicle transport, nuclear transport, and spindle assembly (Gilman 1987; Bourne et al 1991). The GTPases exert their regulatory function through a ‘‘GTPase switch’’ mechanism (Bourne et al 1991) in which the GTPase assumes two alternative conformational states: an active, GTP-bound state and an inactive, GDP-bound state. SRP and SR switch between different functional states (Walter and Johnson 1994; Keenan et al 2001). Upon arrival at the membrane, SRP releases its ‘‘cargo,’’ the RNC, to the translocation apparatus or the translocon (Walter et al 1981; Gilmore et al 1982a, 1982b). Once the RNC is released, both SRP and SR hydrolyze their bound GTPs to drive dissociation of the SRPSR complex, allowing the SRP and SR components to be recycled (Connolly and Gilmore 1989; Connolly et al 1991). Analogous to other GTPases, the switch in the functional states of SRP and SR is coordinated by their GTPase cycles

Methods

Results

Conclusion