Interaction Studies between the Chloroplast Signal Recognition Particle Subunit cpSRP43 and the Full-length Translocase Alb3 Reveal a Membrane-embedded Binding Region in Alb3 Protein

Abstract

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374 -388, and the binding domain within TMD5 was mapped to residues 314 -318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.

Suppressors of the Chloroplast Protein Import Mutant tic40 Reveal a Genetic Link between Protein Import and Thylakoid Biogenesis

“…The cpSRP cooperates in a GTP-dependent manner with cpFtsY, an ortholog of the SRP receptor, to deliver LHCP to ALBINO3 (ALB3), an ortholog of the bacterial YidC protein and a member of the Alb3/Oxa1/YidC insertase family. The ALB3 protein then mediates the insertion (and assembly) of the LHCP protein into the thylakoid membrane (Moore et al, 2000;Falk et al, 2010;Dünschede et al, 2011). Accordingly, Arabidopsis alb3 mutants display a strongly chlorotic/albino phenotype linked to defective LHCP biogenesis (Sundberg et al, 1997;Asakura et al, 2008); by contrast, those with defects affecting a second, paralogous protein called ALB4 appear relatively normal, suggesting that this factor is functionally different (Gerdes et al, 2006;Benz et al, 2009;Trösch et al, 2015).…”

Evolution from the Prokaryotic to the Higher Plant Chloroplast Signal Recognition Particle: The Signal Recognition Particle RNA Is Conserved in Plastids of a Wide Range of Photosynthetic Organisms

“…This heterodimer binds its hydrophobic cargo protein, LHCP, to form the soluble transit complex (Schuenemann et al, 1998). With the participation of the membrane-bound GTPase, cpFtsY, this complex is targeted to the Alb3 translocase in the thylakoid membrane (Moore et al, 2000(Moore et al, , 2003Bals et al, 2010;Lewis et al, 2010;Dünschede et al, 2011).…”

The YidC/Oxa1/Alb3 protein family: common principles and distinct features

“…Two positively charged motifs (AKRS and SKRS) in the C terminus of Alb3 were found to be involved in this interaction (Falk et al , 2010 ), although the requirement for the SKRS motif has been challenged (Dunschede et al , 2011 ). Recently, an additional membrane-embedded cpSRP43 binding region was reported (Dunschede et al , 2011 ), which localizes to a part of TM5 that is oriented to the luminal side of Alb3 and cannot be accessed directly from the stroma.…”

“…The transfer to Alb3 is mediated by the direct interaction of cpSRP43 with the positively charged, stromal-exposed C terminus of Alb3, which is intrinsically disordered and folds upon interaction with cpSRP43 (Tzvetkova -Chevolleau et al, 2007 ;Falk et al , 2010 ;Lewis et al , 2010 ). Two positively charged motifs (AKRS and SKRS) in the C terminus of Alb3 were found to be involved in this interaction (Falk et al , 2010 ), although the requirement for the SKRS motif has been challenged (Dunschede et al , 2011 ). Recently, an additional membrane-embedded cpSRP43 binding region was reported (Dunschede et al , 2011 ), which localizes to a part of TM5 that is oriented to the luminal side of Alb3 and cannot be accessed directly from the stroma.…”

“…Cryo-EM studies with two-dimensional crystals of E. coli YidC (Lotz et al , 2008 ) and with Oxa1 and YidC bound to translating ribosomes (Kohler et al , 2009 ) suggest that YidC forms (head to tail) dimers that are stabilized upon ribosome binding. Native electrophoresis of E. coli YidC (van der Laan et al , 2001 ) and chloroplast Alb3 (Dunschede et al , 2011 ) indicates the presence of monomers and dimers, whereas gel filtration chromatography of the N. crassa Oxa1 suggests that the protein whereas core TMS4 ( E. coli YidC TMS5) cross-links merely at the central and outer membrane face (Klenner and Kuhn , 2012 ). Additional substrate contact surfaces could be located at core TMS3 ( E. coli YidC TMS4) and surprisingly in the noncore TMS1 of E. coli YidC (Klenner et al , 2008 ).…”