Protein targeting into secondary plastids of chlorarachniophytes

Abstract

Most plastid proteins are encoded by the nuclear genome, and consequently, need to be transported into plastids across multiple envelope membranes. In diverse organisms possessing secondary plastids, nuclear-encoded plastid precursor proteins (preproteins) commonly have an N-terminal extension that consists of an endoplasmic reticulum (ER)-targeting signal peptide and a transit peptide-like sequence (TPL). This bipartite targeting peptide is believed to be necessary for targeting the preproteins into the secondary plastids. Here, we newly demonstrate the function of the bipartite targeting peptides of an algal group, chlorarachniophytes, and characterize the functional domains of the TPL in the precursor of a plastid protein, ATP synthase delta subunit (AtpD), using a GFP as a reporter molecule. We show that the C-terminal portion of the TPL is important for targeting the AtpD preprotein from the ER into the chlorarachniophyte plastids, and several positively charged amino acids in the TPL are also necessary for transporting the preprotein across the 2 innermost plastid membranes. Compared with other groups with secondary plastids, the TPL functional domains of the chlorarachniophytes are unique, which might be caused by independent acquisition of their plastids.

Fig. 1.

Confocal images of transformed L. amoebiformis cells, with GFP fluorescence. The images labeled GFP show GFP localization (green), and those labeled plastids show chlorophyll-autofluorescence (red). (A) A cell transformed with pBnAtpD62+GFP showing the GFP localization in the stromas and pyrenoids of plastids. (B) A cell transformed with pGFP-Only (a control), showing the GFP localization in the nucleus and cytoplasm. (C) A cell transformed with pBnAtpD30+GFP, showing the GFP localization in the putative ER. (D) A cell transformed with pLaCRT400+GFP, showing the GFP in the ER. (Scale bar, 5 μm.) DIC, differential interference contrast; Cy, cytoplasm; N, nucleus; PS, plastid stroma; Py, pyrenoid; MP, mature protein.

Fig. 2.

In vivo targeting of GFP fused with BnAtpD precursors possessing various deletions in the TPL. (A) Amino acid sequences of 6 constructs used for the deletion analyses. (A) Dashed line indicates a deleted portion in the TPL of BnAtpD. Red arrowheads indicate the predicted TPL cleavage site. (B) In vivo targeting-efficiencies of GFP fusion proteins, when cells were transformed with each plasmid construct. (C) Confocal images of a cell transformed with pBnAtpD-m6+GFP, showing the GFP localization in the putative ER. (D) Another transformed cell, showing the GFP localization in the possible PPC.

Fig. 3.

In vivo targeting of GFP fused with BnAtpD preproteins possessing various substitutions in the TPL. (A) Amino acid sequences of 12 constructs used for the substitution analyses. Characters in blue indicate the alanine residues substituted at the positions of basic amino acids (red characters). Red arrowheads indicate the predicted TPL cleavage site. (B) In vivo targeting-efficiencies of GFP fusion proteins when cells were transformed with each plasmid construct.

Fig. 4.

Immnocytochemical localization of a GFP fusion protein in the PPC. (A) Confocal images of a L. amoebiformis cell transformed with pBnAtpD-H31-R38A-R50A-R56A-R59A+GFP, showing the GFP localization in the PPC. (Scale bar, 5 μm.) (B) An immunoelectron micrograph of a plastid in a cell transformed with the pBnAtpD-H31A-R38A-R50A-R56A-R59A construct, showing the accumulation of conjugated gold particles (10 nm) in the PPC including a nucleomorph. Nm, nucleomorph.

Fig. 5.

Confirmation of functional compatibility of bipartite plastid-targeting peptides among the chlorarachniophytes, cryptophytes, and apicomplexans. Confocal images of transformed cells expressing heterologous plastid preproteins fused with GFP. (A) Localization of GFP fused with acyl carrier protein of T. gondii (TgAcp183+GFP) in a L. amoebiformis cell. (B) Localization of GFP fused with light harvesting complex protein of G. theta (GtLhcp233+GFP) in a L. amoebiformis cell. (C) Localization of GFP fused with an ATP synthase delta subunit of B. natans (BnAtpD247+GFP) in T. gondii cells. (D) Localization of the GtLhcp233+GFP protein in T. gondii cells. To detect apicoplasts in T. gondii cells, DsRed (red fluorescent protein) fused with an apicoplast-targeted preprotein of T. gondii (TgAcp183+DsRed) was used. AP, apicoplast. (Scale bar, 5 μm.)