GTP Hydrolysis is Not Required for Post‐translational Protein Insertion by a Chloroplast Signal Recognition Particle

Abstract

Light harvesting chlorophyll binding proteins (LHCPs) are a family of nuclear encoded chloroplast thylakoid proteins that are synthesized in the cytosol, imported into the chloroplast stroma, and subsequently directed to the thylakoid membrane. LHCP targeting/insertion into thylakoid membranes requires GTP, a thylakoid protein insertase, and two prokaryote derived GTPases, chloroplast signal recognition particle (cpSRP) and its membrane receptor (cpFtsY). cpSRP binds LHCP in the stroma to form a soluble targeting complex that is directed to thylakoids owing to affinity of cpSRP for cpFtsY, an interaction requiring GTP. LHCP insertion requires Albino‐3 (Alb3), which stimulates GTP hydrolysis by cpSRP/cpFtsY and releases cpSRP from cpFtsY. The role of GTP in LHCP insertion is less clear. Using assays that reconstitute LHCP integration into isolated thylakoids, we have demonstrated that GTP hydrolysis is not required for LHCP insertion. GMP‐PNP, a non‐hydrolysable analogue of GTP, is sufficient to support LHCP integration. Surprisingly, LHCP integration supported by GMP‐PNP, and not GTP, is sensitive to elevated concentrations of free cpSRP/cpFtsY. Our data supports a mechanism in which guanine nucleotide binding by cpSRP/cpFtsY is required for LHCP release from cpSRP whereas GTP hydrolysis serves to recycle cpSRP from cpFtsY for subsequent rounds of targeting. It is hypothesized that guanine nucleotide binding will only serve to release LHCP from cpSRP in the presence of an available Alb3 insertase to prevent unproductive targeting.