A Transit Peptide–Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane

Abstract

Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C 4 species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain-mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition.

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

“…5D, compare lanes 2-4 with 5). Control outer envelope protein, TOC159 (translocon at the outer envelope membrane of chloroplasts, 159 kDa), is known to have an intrinsically thermolysin-resistant portion (77)(78)(79). The resistant 52-kDa fragment was detected when thermolysin was present (Fig.…”

Transit peptide elements mediate selective protein targeting to two different types of chloroplasts in the single-cell C4 species Bienertia sinuspersici

“…In Bienertia, subcellular localization of the main TOC receptors TOC159/132 and TOC34 has been tested previously by in vivo localization studies. However, no preferential accumulation of the fusion constructs in either P- or C-chloroplasts was observed33. Therefore, there is currently no evidence for the involvement of these major TOC complexes in the differential sorting process.…”

“…However, it was later shown that the situation is a little more complex in that different substrates are imported with different efficiencies in chloroplasts of different development stages51. Also in Bienertia, levels of TOC 159 and TOC132 decrease from a very high level in young developing cells to a basal level towards leaf maturation33. Accordingly, either TOC composition or the sorting/import capacity in mature Bienertia protoplasts might be inadequate, especially when fusion constructs are massively overexpressed under the control of the 35 S promoter.…”

Isolation of Chloroplasts from Plant Protoplasts

“…The following protocol was adapted from Smith et al (2003), Yoo et al (2007), and Lung and Chuong (2012).…”

“…Alternatively, protoplasts to be used for transfection experiments should be resuspended in stabilization buffer to a final concentration of 200,000 protoplasts/mL (Lung and Chuong 2012).…”