Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase.
Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.
Kato
A
A
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Hayashi
M
M
Kondo
M
M
Nishimura
M
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism*; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D008830:Microbodies / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014675:Vegetables / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D010940:Plant Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002950:Citrate (si)-Synthase