The Them1 START Domain Regulates Nuclear Trafficking in Brown Adipocytes

Abstract

Thioesterase superfamily member 1 (Them1) is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is a fatty acyl‐CoA thioesterase with tandem hotdog fold thioesterase domains, an N‐terminal set of phosphorylation sites at S15, 18, and 25, and a C‐terminal lipid‐binding steroidogenic acute regulatory protein‐related lipid transfer (START) domain, which modulates thioesterase activity. Cell fractionation studies have demonstrated that Them1 thioesterase activity is localized in microsomes and nuclei, suggesting that it is targeted to specific intracellular compartments. Our aim was to determine the mechanism by which Them1 traffics to the nucleus and its function therein.MethodsIdentification of a putative nuclear localization signal was determined using cNLS mapper ( http://nls‐mapper.iab.keio.ac.jp/cgi‐bin/NLS_Mapper_form.cgi). Full‐length, mutant, or truncated THEM1 constructs were cloned and linked to EGFP. Plasmids or adenoviral vectors were transfected into a differentiated brown adipocyte cell line (MUCAR cells) that does not express Them1. The localization of Them1 was visualized by confocal microscopy via EFGP and mRNA expression was determined using qPCR. Confocal z‐stacks were used to produce 3‐D images with Volocity software. Them1 nuclear localization was evaluated in the presence or absence of the nuclear export inhibitor leptomycin B. Cyclohexamide was used to block new protein synthesis.ResultsPhosphomimic mutations that simulated Them1 phosphorylation at S15 resulted in a diffuse Them1 localization. Stimulation of cells with norepinephrine resulted in Them1 phosphorylation and its reorganization from discrete puncta to a diffuse localization. In the diffuse state, Them1 was transported into the nucleus. Treatment with cyclohexamide suggested that the nuclear Them1 pool was not newly synthesized. We identified a nuclear localization signal in a hinge region located between the 2nd thioesterase domain and the START domain. Deletion of either the nuclear localization signal or the START domain in full length Them 1 blocked nuclear targeting. Conversely, mutant Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus, as evidenced by exit blockade using leptomycin B. Nuclear Them1 regulated mRNA expression levels for a number of genes that regulate lipid metabolism in brown adipocytes.ConclusionsOur results demonstrate that trafficking of Them1 to the nucleus occurs via a nuclear localization signal within the START domain. We speculate that nuclear Them1 regulates the transcription of lipid metabolic enzymes by a mechanism that is distinct from its acyl‐CoA thioesterase activity.Support or Funding InformationThis work was supported by NIH DK103046 to DEC, SJH and EAO.