Analyses in transfected cells and in vitro of a putative peroxisomal targeting signal of rat liver serine:pyruvate aminotransferase.
Serine:pyruvate aminotransferase (SPT; EC 2.6.1.51) of rat liver is a unique enzyme in that it is located in both mitochondria and peroxisomes. To analyze a peroxisomal targeting signal (PTS) of SPT, we constructed in this study various peroxisomal SPT clones having mutations at the C-terminal 20-amino acid region in which a putative PTS is located, and we examined subcellular localization of mutated products expressed in transfected COS-1 cells. When the mutant SPTs were unstable in transfected COS-1 cells, their translocation into peroxisomes was examined using an in vitro peroxisomal import system. Deletion of the C-terminal tripeptide, NKL, and amino acid substitution of K2 (the second lysine from the C-terminus), K4, or E15 abolished or impaired the peroxisomal import of the translated product, resulting in cytosolic accumulation in the cell. In the cases of mutation of R18G, D19A, or K2Q and the conversion to proline of L9, L13, V17, or A20, no products were detected in transfected cells. However, the results of an in vitro peroxisomal import experiment showed that the mutation of L9P, L13P, V17P, and A20P caused loss of the PTS function. When serine was introduced instead of N3 to generate a typical PTS1, the SKL motif, at the C-terminus, all of the proteins having mutations at P5, E11, R12, or E15 showed extensive localization in peroxisomes. These results suggest that the putative C-terminal PTS of SPT is not equivalent to the typical PTS1 shown in acyl-CoA oxidase and urate oxidase, because the PTS of SPT is not restricted to the C-terminal tripeptide. The results also suggest that the alpha-helical structure of the C-terminal region of SPT is important for the stable conformation of the enzyme and the peroxisomal targeting function of its PTS.
Mizuno
Takuji
T
Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan.
Ito
Kouichi
K
Uchida
Chiharu
C
Kitagawa
Masatoshi
M
Ichiyama
Arata
A
Miura
Satoshi
S
Fujita
Kimio
K
Oda
Toshiaki
T
D000818:Animals; D019556:COS Cells; D017931:DNA Primers / Q000737:chemistry; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D020675:Peroxisomes / Q000201:enzymology*; D017354:Point Mutation; D021382:Protein Sorting Signals; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D021382:Protein Sorting Signals; D000637:Transaminases; C022638:serine-pyruvate aminotransferase