The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

Abstract

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.

Interplay between the cpSRP pathway components, the substrate LHCP and the translocase Alb3: An in vivo and in vitro study

“…These data suggested an interaction between cpSRP43 and Alb3 but provided no direct indication of a proteinprotein interaction. Recently, a direct interaction between the recombinant C-terminus of Alb3 and cpSRP43 was proven [13]. However, the physiological role of cpSRP43 in the cpSRP54/ cpFtsY-dependent LHCP transport remains unclear.…”

“…Moore et al [7] reported that cpSRP54 and cpFtsY form the translocation interface together with Alb3 and that complex formation required neither cpSRP43 nor LHCP. However, another study showed that Histagged cpSRP43 copurifies with Alb3 from thylakoid membranes [12], and recently, it was shown that the recombinant C-terminus of Alb3 interacts with cpSRP43 [13].…”

The Intrinsically Disordered N-terminal Region of AtREM1.3 Remorin Protein Mediates Protein-Protein Interactions

“…Examples of ID proteins are the A. thaliana ERD10 and ERD14 dehydrin proteins (48,49), the N-terminal region of the A. thaliana Hy5 bZIP transcription factor involved in photomorphogenesis (50), and the Hordeum vulgare (barley) senescence-associated NAC transcription factor (33). In the case of the Alb3 membrane insertase (51) and the DELLA proteins (34), their ID regions mediate protein interactions.…”

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).…”

“…To address this possibility, we employed in vitro pull-down experiments using recombinant proteins expressed in bacteria ( Figures 6A to 6C). For this work, we used an N-terminally tagged form of the STIC1/ALB4 C terminus (His-ALB4C; residues 345 to 498 of the preprotein), representing most of the stromal C-terminal domain (Falk et al, 2010), and an N-terminally tagged form of the mature STIC2 protein (GST-STIC2; residues 49 to 182 of the preprotein) ( Figure 6A, left side). Equal amounts of soluble lysate from bacteria expressing each protein were mixed together, and then the His-ALB4C protein was affinity purified.…”