Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.
The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.
Chaumont
F
F
Unité de Biochimie Physiologique, University of Louvain, Louvain-la-Neuve, Belgium.
Silva Filho
M de C
Mde C
Thomas
D
D
Leterme
S
S
Boutry
M
M
D000595:Amino Acid Sequence; D001692:Biological Transport; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D005966:Glucuronidase / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D005966:Glucuronidase; D006180:Proton-Translocating ATPases