Protein domains, catalytic activity, and subcellular distribution of neuropathy target esterase in Mammalian cells.
Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, has a predicted amino-terminal transmembrane helix (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Here we describe the expression, in COS cells, of green fluorescent protein-tagged constructs of NTE and mutant proteins lacking the TM or the R- or C-domains. Esterase assays and Western blots of particulate and soluble fractions indicated that neither the TM nor R-domain is essential for NTE catalytic activity but that this activity requires membrane association to which the TM, R-, and C-domains all contribute. Experiments involving proteinase treatment revealed that most of the NTE molecule is exposed on the cytoplasmic face of membranes. In cells expressing a moderate level of NTE and all cells expressing DeltaC-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels of NTE showed aberrant distribution of ER marker proteins and accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in cells expressing DeltaR-NTE or enzymatically inactive S966A-NTE but not DeltaTM-NTE. The data suggest that NTE is anchored in the ER via its TM, that its R- and C-domains also interact with the cytoplasmic face of the ER, and that overexpression of NTE causes ER aggregation via intermolecular association of its C-domains.
Li
Yong
Y
Medical Research Council Toxicology Unit, University of Leicester, United Kingdom. yl8@le.ac.uk
Dinsdale
David
D
Glynn
Paul
P
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002384:Catalysis; D017931:DNA Primers; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D017931:DNA Primers; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase