Plant Physiol. Drug Metab Dispos
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Published on December 29, 2005; 10.1104/pp.105.074575

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Received November 22, 2005
Returned for revision November 25, 2005
Accepted December 1, 2005

Functional characterization of sequence motifs in the transit peptide of Arabidopsis RbcS

Dong Wook Lee , Sookjin Lee , Gil-je Lee , Kwang Hee Lee , Sanguk Kim , Gang-Won Cheong , and Inhwan Hwang *

Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea; Center for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang, 790-784, Korea
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea
Department of Biochemistry, Gyeongsang National University, Jinju, 660-701, Korea

* Corresponding author; email: ihhwang{at}postech.ac.kr.

The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the rubisco complex (RbcS) by examining the ability of various mutant transit peptides to target GFP reporter proteins to chloroplasts in Arabidopsis leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of 8 or 10 amino acids (aa), and generated mutants that had alanine substitutions, or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single or double-T block alanine substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50-100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKEFT in T7. Furthermore, subcellular fractionation revealed that alanine substitution in T1, T3 and T6 produced soluble precursors whereas alanine substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.




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D. W. Lee, J. K. Kim, S. Lee, S. Choi, S. Kim, and I. Hwang
Arabidopsis Nuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs
PLANT CELL, June 1, 2008; 20(6): 1603 - 1622.
[Abstract] [Full Text] [PDF]


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