N‐Terminal Phosphorylation of Thioesterase Superfamily Member 1 (Them1) Regulates its Subcellular Localization in Brown Adipocytes
Authors: Li Yue , Ang Lay‐Hong , Hagen Susan J , Cohen David E 
Published: November 2023

Mammalian cells express acyl‐CoA thioesterases (Acots), which regulate intracellular fatty acid metabolism. Them1, also known as Acot11, suppresses energy expenditure by limiting triglyceride hydrolysis in brown adipose tissue (BAT) and inhibiting the mitochondrial oxidation of lipid droplet (LD)‐derived fatty acids. Them1 comprises tandem N‐terminal thioesterase domains and a C‐terminal fatty acid‐binding domain. This interesting structure suggests that Them1 may act as a “lipid sensor” that controls energy expenditure by blocking access of free fatty acids (FFA) to mitochondria. Proteomics has revealed that there are three tissue‐specific phosphorylation sites near the N‐terminus of Them1 and an in silico analysis of these sites in BAT suggest that protein kinase C‐β (PKCβ) may mediate phosphorylation at these sites.AimThe objective of this study was to determine whether the subcellular localization of Them1 in brown adipocytes supports its putative role in restricting FFA access to mitochondria and whether this effect occurs in response to PKC–mediated phosphorylation of one or more of the serine (S) phosphorylation sites on Them1.MethodsThe full‐length cDNA for Them1 and a series of phosphorylation mutants at S15, S18, and S25 were cloned and linked to EGFP plus an engineered ascorbate peroxidase (Apex2) fusion protein at their C‐termini. Differentiated brown adipocytes (Uldry, Cell Metab, 2006) in culture, which do not constitutively express Them1, were transfected and Them1‐EGFP‐Apex2 was visualized by confocal microscopy (in living cells) or by diaminobenzidine development and electron microscopy. Volocity software was used to produce 3‐D images from confocal z‐stacks. To explore the effect of phosphorylation on the localization of Them1, phorbol 12‐myristate 13‐acetate (PMA) was applied to activate endogenous PKC in brown adipocytes.ResultsThem1‐EGPF‐Apex2 formed small, punctate structures near LD, especially LD with a diameter of less than 5 μm. 3‐D images revealed that Them1 is sandwiched between mitochondria and LD that were closely associated with one another. A short N‐terminal 36‐residue deletion or phosphomimetic mutation of S15 alone (S15D) was sufficient to inhibit Them1 interactions, resulting in a diffuse distribution of the protein within the cytoplasm. In response to PMA treatment, Them1 was redistributed to a punctate pattern, consistent with LD/mitochondria associations. The Them1 construct containing a S15A mutation did not redistribute in response to PMA.ConclusionsThe subcellular localization of Them1 is largely attributable to PKC‐mediated phosphorylation of the N‐terminus, particularly at the S15 site. Them1 specifically localizes between LD and mitochondria in living cells, indicating that it is well positioned to control energy expenditure by limiting the access of fatty acids to mitochondria.Support or Funding InformationFunding was from the National Institutes of Health, R01DK103046 (SJH and DEC).