A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

Abstract

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. (3); the N-terminal portion governs transport across the envelope, whereas the C-terminal portion directs translocation across the thylakoid membrane (4). In the case of LHCP, the mature apoprotein contains sufficient information for thylakoid targeting (5), although the specific signal has not yet been identified (6, 7). Studies of the early events of LHCP trafficking using isolated chloroplasts showed that LHCP is present as a soluble form in the stroma prior to integration into thylakoids (8). When integration was inhibited, this intermediate LHCP accumulated in the stroma as a larger complex (9). Formation of this complex, which we have designated the "transit complex," maintains LHCP solubility and integration competence (9). Both transit complex formation and LHCP integrationThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.into thylakoids have been reconstituted in vitro; both require a proteinaceous component(s) of the stroma (9, 10).The chaperone nature of this "stromal factor" led to the suggestions that heat shock proteins Cpn6O (11) and Hsp7O (12) were involved in LHCP integration. These molecular chaperones are abundant in the stroma (13,14), are able to bind polypeptides and release them upon ATP hydrolysis, and have been shown to be involved in mitochondrial protein import (15). However, immunoprecipitation experiments with antibodies against the two proteins failed to reveal the presence of Cpn6O and Hsp7O in the transit complex (9, 14). Furthermore, stromal extract depleted of Hsp7O still supported transit complex formation and LHCP integration, indicating that neither process requires this chaperone (14). The recent demonstration of a stromal GTP requirement (16) for LHCP integration now suggests the involvement of a guanine nucleotide-binding protein in this process. One likely candidate is 54CP, a chloroplast homologue of the 54-kDa protein of the mammalian signal recognition particle (SRP) (17). Protein transport across or integration into the endoplasmic reticulum (ER) is mediated by a GTP-dependent mechanism involving SRP (18). The 54-kDa polypeptide subunit of SRP (SRP54) plays a major role in SRP-dependent targeting. It binds guanine nucleotides as well as nascent polypeptides that are destined for ER transport or integration (19). Furthermore, SRP54 appears to be required for docking to the SRP receptor (20) and hydrolyzes the GTP required for dissociation of SRP from the receptor (21). In this report, we show that 54CP is bound to LHCP in the transit complex and is essential for LHCP integration into the thylakoid membrane. (p) were prepared by in vitro transcription and translation as described (7). Unlabeled pLHCP and pOE33 (33-kDa subunit of the oxygenevolving complex) were expressed in Escherichia coli and pur...

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

“…The identification of LTD in LHCP import and subsequent delivery to the cpSRP pathway was unexpected, as both the envelope and cpSRP transport pathways have been extensively investigated and this component has not been detected before 11,15,20,21,49,50 . One possibility is that the inability of LTD to form the stable complexes with cpSRP43 or with Tic40 and Tic110, making it difficult to be detected using the biochemical assays currently applied in the identification of the components of the envelope and cpSRP transport pathways 11,15,20,21,49,50 .…”

“…One possibility is that the inability of LTD to form the stable complexes with cpSRP43 or with Tic40 and Tic110, making it difficult to be detected using the biochemical assays currently applied in the identification of the components of the envelope and cpSRP transport pathways 11,15,20,21,49,50 . Another possibility is that an intermediate stage of such process cannot be trapped or reconstituted in vitro for further identification of the components involved in because the transport from the inner envelop to the SRP pathway is so rapid.…”

“…Moreover, LTD cannot be identified by analogue with protein export pathway in mammalian and Escherichia coli. Homologues of prokaryotic SRP54 and its receptor FtsY have been identified in chloroplasts 15,20 . However, LTD and cpSRP43 homologues are found in higher plants, moss and green algae but not in cyanobacteria and red algae 23 ( Supplementary Fig.…”

“…LHCP form a soluble transit complex with cpSRP, which consists of cpSRP54 and cpSRP43, after import into the chloroplast [15][16][17] . The ankyrin domain of cpSRP43 interacts with a conserved L18 region between the second and third transmembrane domains of LHCP 18,19 .…”

Efficient Interaction between Two GTPases Allows the Chloroplast SRP Pathway to Bypass the Requirement for an SRP RNA

“…cpSRP54 and cpFtsY are the chloroplast homologues of SRP and SR GTPases, respectively (Franklin and Hoffman, 1993;Li et al, 1995;Tu et al, 1999). cpSRP54 recognizes its cargo, the light-harvesting chlorophyll-binding proteins (LHCP), via a protein adaptor cpSRP43 (Tu et al, 2000).…”