Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor.
Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.
Kamigaki
Akane
A
Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8528, Japan.
Mano
Shoji
S
Terauchi
Kaori
K
Nishi
Yukiko
Y
Tachibe-Kinoshita
Yuki
Y
Nito
Kazumasa
K
Kondo
Maki
M
Hayashi
Makoto
M
Nishimura
Mikio
M
Esaka
Muneharu
M
D001483:Base Sequence; D001665:Binding Sites; D002374:Catalase / Q000235:genetics / Q000378:metabolism*; D028464:Cucurbita / Q000201:enzymology / Q000502:physiology*; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000235:genetics / Q000502:physiology*; D010940:Plant Proteins / Q000502:physiology; D030821:Plants, Genetically Modified; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000502:physiology
D017931:DNA Primers; D008164:Luminescent Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D010940:Plant Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002374:Catalase