The C Terminus of a Chloroplast Precursor Modulates Its Interaction with the Translocation Apparatus and PIRAC

Abstract

The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (⌬52, ⌬67, and ⌬74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of 35 S-prSSU. Both IC 50 and K i values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and ⌬52 were properly processed and accumulated within the chloroplast, whereas ⌬67 and ⌬74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.

In Vitro Comparative Kinetic Analysis of the Chloroplast Toc GTPases

“…Additionally, the use of short synthetic peptides could have unintentionally split a continuous recognition element present in the full-length transit peptide. Finally, short synthetic peptides would abrogate any structural contributions from the mature domain as has been clearly demonstrated for prSSU (61).…”

Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

“…The data from mSSU, SSR, and FDR were fitted to straight lines. Determinants of Transit Peptide Recognitionby a shift in size from a larger precursor to a smaller TP-devoid mature protein ( Figure 4D) (Friedman and Keegstra, 1989;Dabney-Smith et al, 1999). The import competitions can be performed by titrating competitors to a constant concentration of 35 S-prSSU.…”

“…This intermediate can be observed using radioactivity or by various fluorescence assays, such as flow cytometry or confocal microscopy, that permit the quantification and imaging of the bound precursor (Subramanian et al, 2001). In the past, we employed quantitative import competition assays that used competitive inhibitors to determine specific inhibitory values of the import process, such as the inhibitor constant (K i ) and the half maximal inhibitory concentration (IC 50 ) (Dabney-Smith et al, 1999). Here, we used the competition assays to evaluate the binding of the radiolabeled precursor of small subunit of Rubisco ( 35 S-prSSU).…”

“…The import assays were performed using a modified method from Dabney-Smith et al (1999). Briefly, the competition assays were performed in a 300-mL reaction containing 0.125 mg chlorophyll/mL chloroplasts, 100 nM 35 S-prSSU, 1 mM BME, 2 mM Mg-ATP, 0.5% BSA, 250 mM urea, 330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0, and various amounts of competitor proteins/ TPs.…”

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

“…The import assays were performed using a modified method from Dabney-Smith et al (34). Briefly, the assays were performed in a 500-l reaction containing 0.25 mg of chlorophyll/ml chloroplasts, labeled protein, 2 mM L-Met, 10 mM DTT, 2 mM Mg-ATP, 0.5% BSA, 300 mM urea, 330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0.…”