The α‐helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP⅐LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.Mitochondrial inner membranes and chloroplast thylakoid membranes are densely populated with protein complexes vital to the production of metabolic energy. For both membrane systems, biogenesis requires specialized protein sorting and integration systems, which localize nucleus-and organelle-encoded proteins to the target membrane. Consistent with the prokaryotic origin of mitochondria and chloroplasts, protein insertion into their energy-generating membranes is accomplished via the action of Oxa1p and Albino3 (Alb3), respectively, which belong to a family of protein insertases that includes YidC in bacteria (1-6).Although YidC/Oxa1p/Alb3 homologues vary dramatically in length (225-795 residues), all share a conserved hydrophobic core of about 200 residues (2) that extends across five transmembrane domains leaving the C terminus exposed to the cytoplasm, matrix, or stroma, respectively. Complementation studies demonstrated that the core regions of both Oxa1p and Alb3 functionally replace the core of YidC to insert membrane proteins via a "YidC only" pathway (7,8). Similarly, a chimera of YidC fused with the C-terminal ribosome-binding domain of Oxa1p was useful in demonstrating that the core region of YidC can functionally replace the core region of Oxa1p (9). These experimental results show that the core regions of YidC/ Oxa1p/Alb3 are at least partially interchangeable and house the capacity for assisting membrane protein transition into adjacent bilayers. They also support the possibility that a conserved function of the YidC/Oxa1p/Alb3 C terminus is to bind soluble targeting machinery. For example, the hydrophilic C-terminal extension of Oxa1p forms an ␣-helical domain essential for interacting with the ribosome during cotranslational integration (10, 11). Like Oxa1p, Alb3 contains a hydrophilic C-terminal extension that may play a critical role in protein targeting (12, 13). Alb3 works in conjunction with a post-translational chloroplast signal recognition particle (cpSRP)...

mentioning

confidence: 85%

Section: Cpsrp43 Mediates Alb3 Regulation Of Cpsrpmentioning

confidence: 99%

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components*

Lewis

Marty

Kathir

et al. 2010

“…Third, apart from acting as a chaperone, At-cpSRP43 can function as a targeting factor because it is able to interact directly with the translocase Alb3 within the thylakoid membrane (19 -22). In higher plants, the complete pool of stromal cpSRP43 is associated with cpSRP54, forming a stable cpSRP complex (7,8) the subunits of which interact with high affinity as a K d value of 2.5 nM was described for the interaction between At-cpSRP43 and At-cpSRP54M (32). Likewise, the ability of the cpSRP proteins from the moss Physcomitrella to form stable complexes at least in vitro has been recently described (25).…”

Section: Discussionmentioning

confidence: 99%

Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution

Dünschede

Träger

Schröder

et al. 2015

Self Cite

Background: In chloroplasts of higher plants, a heterodimeric cpSRP43⅐cpSRP54 complex targets LHC proteins to the thylakoid membrane. Results: In green algae, cpSRP43 alone forms a targeting complex with LHC proteins. Conclusion: The coevolution of LHC proteins and cpSRP43 occurred independently of complex formation with cpSRP54. Significance: The results provide new insights into the evolution of cpSRP-dependent protein transport.

“…The cpSRP43 Chromodomains Can Bind Alb3 and cpSRP54 Simultaneously-It was previously shown that cpSRP43 CD2 is necessary and sufficient to bind the cpSRP54 M-domain (15,34,35). A stretch of 10 amino acids (RRKR peptide, supplemental Fig.…”

Section: The Alb3-cpsrp43 Interaction Requires Two Positively Chargedmentioning

confidence: 99%

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

Falk

Ravaud

Koch

et al. 2010

103

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.