A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Abstract

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5-to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication. INTRODUCTIONThe signal recognition particle (SRP) and the SRP receptor (SR) comprise the major cellular machineries that cotranslationally deliver newly synthesized proteins from the cytosol to target membranes (Walter and Johnson, 1994;Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991;Gorlich et al., 1992;Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP • SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keen...

Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

“…As shown in previous work and in the preceding paper (51), the M-domain of cpSRP54 interacts with conserved basic residues in the cpFtsY G-domain; this interaction is required for rapid assembly of a stable cpSRP54⅐cpFtsY complex that was previously observed only when an artificial stimulant, Nikkol, was used (28). Intriguingly, here we found that both the liposome-induced stimulation of the cpSRP54⅐cpFtsY complex assembly and the Alb3-mediated regulation of GTP hydrolysis in this complex also require the M-domain of cpSRP54.…”

“…As described in the accompanying paper (51), the cpSRP54 M-domain provides a platform to coordinate rapid and stable complex assembly between cpSRP54 and cpFtsY, thus replacing the otherwise conserved SRP RNA (28). Interestingly, deletion of the cpSRP54 M-domain abolished the stimulatory effect of PG on complex assembly (Fig.…”

“…The otherwise universally conserved SRP RNA is no longer present; in our previous work and our accompanying paper (51), we described how the M-domain of cpSRP54 replaces the SRP RNA to enable efficient interaction between the cpSRP54 and cpFtsY GTPases (25)(26)(27)(28)(29). In addition, the transition to a post-translational mode of targeting necessitated the evolution of a novel SRP subunit, cpSRP43, which binds with high affinity and specificity to LHCPs and provides an effective chaperone that prevents these multipass integral membrane proteins from aggregation (30,31).…”

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

“…The finding that the preformed closed conformation of cpFtsY is not phylogenetically correlated to the loss of the cpSRP RNA indicates that this conformational change of cpFtsY is not sufficient to enable an efficient cpFtsY/cpSRP54 complex formation. Interestingly, it has been shown that in Arabidopsis the M-domain of cpSRP54 mimics the function of the SRP RNA by providing a significant acceleration of complex formation between the cpSRP GTPases (Jaru- Ampornpan et al, 2009). This leads to the speculation that the M-domain of P. patens cpSRP54 might still resemble the classic bacterial M-domain, which has no stimulating influence on complex formation (Jaru-Ampornpan et al, 2009) and might therefore still depend on an SRP RNA for efficient cpFtsY binding.…”

“…It was suggested that this modified position of the Asp residue in cpFtsY contributes to the significantly higher nucleotide specificity compared with prokaryotic FtsY (Jaru-Ampornpan et al, 2007;Chandrasekar et al, 2008). Besides the closed conformation of cpFtsY, the M-domain of cpSRP54 was shown to play an important role for an efficient interaction between the cpSRP GTPases by stimulating complex formation (Jaru-Ampornpan et al, 2009). …”