Chloroplast import signals: the length requirement for translocation in vitro and in vivo.
Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids-longer than the transit peptide length of many experimentally confirmed chloroplast proteins-are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.
Bionda
Tihana
T
Molecular Cell Biology of Plants, Department of Biosciences, Center for Membrane Proteomics and Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Max von Laue Strasse 9/N200/R3.02, Frankfurt, Germany.
Tillmann
Bodo
B
Simm
Stefan
S
Beilstein
Kim
K
Ruprecht
Maike
M
Schleiff
Enrico
E
D000595:Amino Acid Sequence; D000818:Animals; D002736:Chloroplasts / Q000378:metabolism*; D064211:Connectin; D007136:Immunoglobulins / Q000737:chemistry; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D015398:Signal Transduction
D064211:Connectin; D007136:Immunoglobulins; D009124:Muscle Proteins; D010455:Peptides; D010940:Plant Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases