Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

Zht Cheng Wu,Jeanine De Keyzer,Alexej Kedrov,Arnold J.M Driessen

doi:10.1074/jbc.m111.297911

Zht Cheng Wu, Jeanine De Keyzer + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.297911

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Abstract

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

Highlights

Both SecA and the ribosome need to interact with the translocon during membrane protein insertion
To correct for “bulk” contributions to the Surface Plasmon Resonance (SPR) signal and nonspecific binding to the membrane and chip surface, all measurements were corrected for binding to IMVs bearing endogenous SecYEG levels that were immobilized in a reference channel [26]
To validate that the observed SPR response reflected the binding of ribosomes to the SecYEG complex, we analyzed the interaction between ribosomes and two SecY mutant complexes, i.e. SecY(R255E,R256E)EG and SecY(R357E)EG

Summary

Background

Both SecA and the ribosome need to interact with the translocon during membrane protein insertion. During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Structural, biochemical, and computational approaches have provided detailed insights in the interaction between SecYEG and its cytosolic binding partners Both SecA and the ribosome interact primarily with the largest subunit of the SecYEG complex, i.e. SecY. Many substrates of the translocon only require the action of either SecA or the ribosome for translocation or membrane insertion, the biogenesis of membrane proteins with large periplasmic loops or domains, such as FtsQ and CyoA, requires both functions [15, 16]. Our data demonstrate that SecA and the ribosome compete for binding to the SecYEG complex and that substrate loading is an important determining factor in this process

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION