In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts.
The N-terminal transit peptide of chloroplast proteins is necessary and sufficient to direct proteins to the chloroplasts. However, the requirement of the transit peptide of chloroplast proteins is not fully understood. In this study we investigated the requirement of a transit peptide at the level of amino acid sequence using an in vivo targeting approach. Targeting experiments with green fluorescent protein (GFP) fusion proteins containing varying lengths of the N-terminal region of the small subunit of rubisco complex (RbcS) revealed that at least 73 amino acid residues of the N-terminal region is required to direct GFP to the chloroplasts without affecting the efficiency. Even a small deletion from the C- or N-termini of the minimal length of the transit peptide results in strong inhibition of targeting. Also, a small internal deletion within the minimal transit peptide strongly affected targeting of GFP fusion proteins. However, when we replaced one or two amino acid residues of the transit peptide with corresponding numbers of alanine residues sequentially, all the mutants were imported into chloroplasts with 80 to 100% efficiency. Together these results suggest that the overall context of amino acid sequence, but not any specific amino acid residue, of the transit peptide is critical for targeting to the chloroplasts.
Lee
Kwang Hee
KH
Center for Plant Intracellular Trafficking and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
Kim
Dae Heon
DH
Lee
Sang Woo
SW
Kim
Zhoo Hyeon
ZH
Hwang
Inhwan
I
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000502:physiology; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics* / Q000502:physiology; D021381:Protein Transport; D011523:Protoplasts / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D012273:Ribulose-Bisphosphate Carboxylase; D000409:Alanine