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Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

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Tahay, Gaëlle [UCL] Wiame, Elsa [UCL] Tyteca, Donatienne [UCL] Courtoy, Pierre J. [UCL] Van Schaftingen, Emile [UCL]

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.

Document type Article de périodique (Journal article) – Article de recherche – Journal Article, Research Support, Non-U.S. Gov't
Access type Accès restreint
Publication date 2012
Language Anglais
Journal information "Biochemical Journal" - Vol. 441, no. 1, p. 105-112 (2012)
Peer reviewed yes
Publisher Portland Press Ltd. ((United Kingdom) London)
issn 0264-6021
e-issn 1470-8728
Publication status Publié
Affiliations UCL - SSS/DDUV - Institut de Duve
UCL - (SLuc) Service d'hématologie
MESH Subject Protein Transport ; Protein Conformation ; Point Mutation ; Molecular Sequence Data ; Humans ; HEK293 Cells ; Endoplasmic Reticulum ; Cricetinae ; Cell Membrane ; CHO Cells ; Animals ; Amino Acid Sequence ; Acetyltransferases - genetics - metabolism
Links
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Bibliographic reference Tahay, Gaëlle ; Wiame, Elsa ; Tyteca, Donatienne ; Courtoy, Pierre J. ; Van Schaftingen, Emile. Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase. In: Biochemical Journal, Vol. 441, no. 1, p. 105-112 (2012)
Permanent URL http://hdl.handle.net/2078/105235