FIT2 is a lipid phosphate phosphatase crucial for endoplasmic reticulum homeostasis

Abstract

These authors contributed equally.Author contributions: MB, TW, and RF conceived the project. MB acquired and analyzed the data. NM did the image analysis of LD formation, and bioinformatic analyses of lipidomics data. SDE helped in the establishment of stable cell lines and did LLSM acquisition. SB developed lipidomics pipeline and analyzed the data. LB helped in the enzymatic activities, protein purification and did qPCR analysis. MC did endoplasmic reticulum features quantification. XNL and MMG did EM. MB, TW, and RF wrote the paper.Running title: FIT2 is an ER-localized lipid phosphate phosphatase SUMMARYThe endoplasmic reticulum (ER) protein Fat-Induced Transcript 2 (FIT2) has emerged as a key factor in lipid droplet (LD) formation, although its molecular function is unknown.Highlighting its importance, FIT2 orthologs are essential in worms and mice, and FIT2 deficiency causes a deafness/dystonia syndrome in humans. Here we show that FIT2 is a lipid phosphate phosphatase (LPP) enzyme that is required for maintaining the normal structure of the ER. Recombinant FIT2 exhibits LPP activity in vitro and loss of this activity in cells leads to ER membrane morphological changes and ER stress. Defects in LD formation in FIT2 depletion appear to be secondary to membrane lipid abnormalities, possibly due to alterations in phospholipids required for coating forming LDs. Our findings uncover an enzymatic role for FIT2 in ER lipid metabolism that is crucial for ER membrane homeostasis.

Yeast FIT2 homolog is necessary to maintain cellular proteostasis by regulating lipid homeostasis

“…Conversely, it was found that the deletion of FIT2 greatly compromised LD formation in the model organisms, Danio rerio and Caenorhabditis elegans, as well as in the pathogenic yeast Candida parapsilosis (12,17,18). Similar findings were observed in FIT2-ablated human cells cultured in vitro (19). All these reports further reinforced the initial hypothesis of the FIT proteins directly functioning in LD biogenesis.…”

“…In-depth analysis of the ScFIT protein sequences have since then revealed the presence of the catalytic site of a lipid phosphatase (22). This finding was further corroborated by a separate study that reported the same observation on the mammalian FIT2 sequence (19). In vitro analyses have identified the capacity of mammalian FIT2 to hydrolyze phosphates from phosphatidic acid (PA) and lyso-PA to yield diacylglycerol (DAG) and monoacylglycerol (MAG), respectively.…”

“…In contrast, S. cerevisiae deletion mutants for either or both ScFIT genes retain their capacity to form LDs, with size and number comparable to WT (14,19). Upon closer investigation, it was found that LDs fail to completely bud off from the ER in the absence of the ScFIT proteins (17), presumably through alterations in ER membrane lipid properties (20).…”

The assembly of lipid droplets and their roles in challenged cells

“…We confused the Hayes reference with a publicly available preprint in which weak lipid phosphatase activity was reported (preprint: Becuwe et al, 2018). In this reference, overexpression of either of the two yeast FIT2 proteins in a strain deficient in the three known phosphatidic acid phosphatases yielded no significant PA phosphatase activity.…”

“…In this reference, overexpression of either of the two yeast FIT2 proteins in a strain deficient in the three known phosphatidic acid phosphatases yielded no significant PA phosphatase activity. We confused the Hayes reference with a publicly available preprint in which weak lipid phosphatase activity was reported (preprint: Becuwe et al, 2018).…”