No abstract

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP͞LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts. Signal recognition particle (SRP) and its receptor comprise essential components of a signal peptide-based protein targeting mechanism that is conserved across evolutionary boundaries (1-3). SRPs in the cytosols of eukaryotes and Escherichia coli target proteins cotranslationally to the endoplasmic reticulum and cytoplasmic membrane, respectively. Targeting is initiated as a result of SRP binding to the hydrophobic domain of amino-terminal signal peptides or signal anchors as they emerge from the ribosome. The entire ribosome͞nascent polypeptide chain complex (RNC) then is piloted by SRP to an SRP receptor that functions at the membrane. GTP binding and hydrolysis by SRP and its receptor result in both the release of SRP from its receptor and the release of SRP from the RNC, whereupon the nascent chain enters a translocation pore that directs the translating polypeptide into or across the lipid bilayer.An organellar SRP, which exhibits striking structural and functional differences from cytosolic SRPs, also has been identified in chloroplasts (4, 5). Chloroplast SRP (cpSRP) is a soluble Ϸ200-kDa stromal particle that contains an evolutionary conserved 54-kDa subunit (cpSRP54) as well as a unique 43-kDa polypeptide (cpSRP43) (6). Unlike cytosolic SRPs, an RNA moiety is conspicuously lacking in cpSRP. Biochemical and genetic evidence have demonstrated that cpSRP functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins belonging to the chlorophyll...

No abstract

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.