Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1.
Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.
Hirano
Tohko
T
Department of Biochemistry, Gunma University Graduate School of Medicine, Japan.
Kishi
Mikiko
M
Sugimoto
Hiroyuki
H
Taguchi
Ryo
R
Obinata
Hideru
H
Ohshima
Noriyasu
N
Tatei
Kazuaki
K
Izumi
Takashi
T
D002460:Cell Line; D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000378:metabolism; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D021381:Protein Transport; D013347:Subcellular Fractions / Q000201:enzymology; D013869:Thiolester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*
D008244:Lysophosphatidylcholines; D050505:Mutant Proteins; D010455:Peptides; D008245:Lysophospholipase; C402226:LYPLA1 protein, human; D013869:Thiolester Hydrolases