The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

Kim

Journal of Biological Chemistry

et al. 2003

Toc159, a protein located in the outer envelope membrane and the cytosol, is an important component of the receptor complex for nuclear-encoded chloroplast proteins. We investigated the molecular mechanism of protein import into chloroplasts by atToc159 using the ppi2 mutant, which has a T-DNA insertion at atToc159, shows an albino phenotype, and does not survive beyond the seedling stage due to a defect in protein import into chloroplasts. First we established that transiently expressing atToc159 in protoplasts obtained from the white leaf tissues of ppi2 plants complements the protein import defect into chloroplasts. Using this transient expression approach and a series of deletion mutants, we demonstrated that the C-terminal membrane-anchored (M) domain is targeted to the chloroplast envelope membrane in ppi2 protoplasts, and is sufficient to complement the defect in protein import. The middle GTPase (G) domain plays an additional critical role in protein import: the atToc159[S/N] and atToc159[D/L]mutants, which have a mutation at the first and second GTP-binding motifs, respectively, do not support protein import into chloroplasts. Leaf cells of transgenic plants expressing the M domain in a ppi2 background contained nearly fully developed chloroplasts with respect to size and density of thylakoid membranes, and displayed about half as much chlorophyll as wild-type cells. In transgenic plants, the isolated M domain localized to the envelope membrane of chloroplasts but not the cytosol. Based on these results, we propose that the M domain is the minimal structure required to support protein import into chloroplasts, while the G domain plays a regulatory role.

Section: Transient Expression Of Attoc159 Supports Protein Import Intmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The M Domain of atToc159 Plays an Essential Role in the Import of Proteins into Chloroplasts and Chloroplast Biogenesis

Kim

Journal of Biological Chemistry

et al. 2003

“…The transit peptides of LHCPs, however, comprise only the envelope transit sequences, which target the hydrophobic proteins to the stroma, where they are able to insert into the thylakoid membrane in the absence of their transit peptides (Lamppa, 1988). Thus, the information for LHCP integration into the thylakoid resides in the mature protein (Hand et al, 1989). Attempts at localizing the targeting information have not been successful (Auchincloss et al, 1992;Huang et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

A Chromodomain Protein Encoded by the Arabidopsis CAO Gene Is a Plant-Specific Component of the Chloroplast Signal Recognition Particle Pathway That Is Involved in LHCP Targeting

et al. 1999

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO ), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos ) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes. INTRODUCTIONHigher plants require chloroplasts for essential functions ranging from photosynthesis to sugar, lipid, and amino acid biosyntheses. Many of the proteins required for the light reactions of photosynthesis, including the chlorophyll-containing antenna proteins, are localized in the thylakoid membrane. Thylakoid proteins are encoded by both the nuclear and chloroplast genomes. Nuclear-encoded proteins destined for the thylakoid membrane are synthesized with a cleavable N-terminal extension (transit peptide) that targets the protein across the envelope membranes into the stroma (reviewed in Cline and Henry, 1996;Kermode, 1996;Lübeck et al., 1997). Multiple mechanisms exist for targeting proteins from the stroma to the thylakoid membrane (reviewed in Cline and Henry, 1996;Klösgen, 1997). These include spontaneous insertion, a chloroplast Sec r -dependent pathway, a chloroplast signal recognition particle (cpSRP) pathway, and a pathway requiring only an electrochemical potential of ⌬ pH across the thylakoid membrane. These pathways can be distinguished by specific requirements for stromal proteins and/or by energetic requirements when in 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed: E-mail lnussaume @cea.fr; fax 33-442-25-4656. 88The Plant Cell vitro reconstitution assays are performed. In vitro, precursor proteins are targeted exclusively via one of the aforementioned pathways.LHCIIb proteins are the most abundant of the thylakoid membrane ...

“…Intra-organellar routing which depends exclusively on the mature polypeptide and apparently does not require the transit peptide is a mechanism which is shared by various thylakoid membrane proteins. It was first described for LHCP ll (Hand et al, 1989;Lamppa, 1988; Viitanen et a/., 1988), but proteins which are not constituents of the light-harvesting apparatus, like the Rieske Fe/S protein, a membrane-attached, lumenal subunit of the cytochrome bdfcomplex, can also assemble with the membrane independent of their transit peptides (Bartling et a/., 1990). However, this widespread mechanism is not obligatory to all thylakoid membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

The 20 kDa apoprotein of the CP24 complex of photosystem II: An alternative model to study import and intra‐organellar routing of nuclear‐encoded thylakoid proteins

1993

SummaryThe 20 kDa polypeptide, the apoprotein of the chlorophyll a/b antenna complex CP24 associated with photosystem II, is a remote relative of light-harvesting complex (LHC) apoproteins and thus a member of the extended cab gene family. LHC apoproteins are polytopic integral components of the thylakoid membrane with probably three transmembrane segments which originate in nuclear genes and are made in the cytosol as precursors. They possess exclusively stromatargeting transit peptides for import into the organelle and integrate into the thylakoid membrane via uncleaved hydrophobic domains of the mature protein, The CP24 apoprotein displays intriguing structural differences to LHC apoproteins with a potential impact on the routing and targeting processes during biogenesis. In particular, it lacks a pronounced second hydrophobic segment in the mature polypeptide chain found in LHCPs, and carries a transit peptide that is reminiscent of thylakoid-targeting transit peptides. We have used in organello assays with isolated intact chloroplasts and the authentic precursor of the 20 kDa apoprotein from spinach, or appropriate chimaeric polypeptides consisting of a transit peptide and the mature part of various nuclear-encoded thylakoid proteins of known location and targeting epitopes, in order to resolve the characteristics of its targeting properties, as well as t o determine the contribution of the individual parts of the precursor molecule to its import and subsequent intra-organellar routing. Our experiments demonstrate that the transit peptide of the CP24 apoprotein is required only for the import of the protein into the organelle. All subsequent Steps, such as the integration of the protein into the thylakoid membrane, binding of chlorophyll, assembly into the cp24 complex and migration to the grana lamellae, still take place if the authentic transit peptide is re-