How proteins specifically localize to the phospholipid monolayer surface of lipid droplets (LDs) is being unraveled. We review here the major known pathways of protein targeting to LDs and suggest a ...classification framework based on the localization origin for the protein. Class I proteins often have a membrane-embedded, hydrophobic ‘hairpin’ motif, and access LDs from the endoplasmic reticulum (ER) either during LD formation or after formation via ER–LD membrane bridges. Class II proteins access the LD surface from the cytosol and bind through amphipathic helices or other hydrophobic domains. Other proteins require lipid modifications or protein–protein interactions to bind to LDs. We summarize knowledge for targeting and removal of the different classes, and highlight areas needing investigation.
One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is ...the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.
A variant (148M) in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) is a major risk factor for fatty liver disease. Despite its clinical importance, the pathogenic mechanism linking ...the variant to liver disease remains poorly defined. Previously, we showed that PNPLA3(148M) accumulates to high levels on hepatic lipid droplets (LDs). Here we examined the effect of that accumulation on triglyceride (TG) hydrolysis by adipose triglyceride lipase (ATGL), the major lipase in the liver. As expected, overexpression of ATGL in cultured hepatoma (HuH‐7) cells depleted the cells of LDs, but unexpectedly, co‐expression of PNPLA3(wild type WT or 148M) with ATGL inhibited that depletion. The inhibitory effect of PNPLA3 was not caused by the displacement of ATGL from LDs. We tested the hypothesis that PNPLA3 interferes with ATGL activity by interacting with its cofactor, comparative gene identification‐58 (CGI‐58). Evidence supporting such an interaction came from two findings. First, co‐expression of PNPLA3 and CGI‐58 resulted in LD depletion in cultured cells, but expression of PNPLA3 alone did not. Second, PNPLA3 failed to localize to hepatic LDs in liver‐specific Cgi‐58 knockout (KO) mice. Moreover, overexpression of PNPLA3(148M) increased hepatic TG levels in WT, but not in Cgi‐58 KO mice. Thus, the pro‐steatotic effects of PNPLA3 required the presence of CGI‐58. Co‐immunoprecipitation and pulldown experiments in livers of mice and in vitro using purified proteins provided evidence that PNPLA3 and CGI‐58 can interact directly. Conclusion: Taken together, these findings are consistent with a model in which PNPLA3(148M) promotes steatosis by CGI‐58‐dependent inhibition of ATGL on LDs.
The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. ...Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.
Cytosolic lipid droplets (LDs) are the main storage organelles for metabolic energy in most cells. They are unusual organelles that are bounded by a phospholipid monolayer and specific surface ...proteins, including key enzymes of lipid and energy metabolism. Proteins targeting LDs from the cytoplasm often contain amphipathic helices, but how they bind to LDs is not well understood. Combining computer simulations with experimental studies in vitro and in cells, we uncover a general mechanism for targeting of cytosolic proteins to LDs: large hydrophobic residues of amphipathic helices detect and bind to large, persistent membrane packing defects that are unique to the LD surface. Surprisingly, amphipathic helices with large hydrophobic residues from many different proteins are capable of binding to LDs. This suggests that LD protein composition is additionally determined by mechanisms that selectively prevent proteins from binding LDs, such as macromolecular crowding at the LD surface.
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•Lipid droplet surfaces are characterized by phospholipid packing defects•Packing defects recruit amphipathic helices to the lipid droplet surface•Large, hydrophobic residues of amphipathic helices initially bind packing defects•In isolation, many amphipathic helices accumulate on lipid droplets
Prévost, Sharp, et al. explore how cytosolic proteins target the surface of lipid droplets (LDs). A distinctive surface structure makes LDs suited to recruiting large hydrophobic amino acid-enriched protein motifs. Rather than specific LD protein-targeting mechanisms, regulation is likely at the level of preventing promiscuous association by non-LD proteins.
Mitochondria undergo architectural/functional changes in response to metabolic inputs. How this process is regulated in physiological feeding/fasting states remains unclear. Here we show that ...mitochondrial dynamics (notably fission and mitophagy) and biogenesis are transcriptional targets of the circadian regulator Bmal1 in mouse liver and exhibit a metabolic rhythm in sync with diurnal bioenergetic demands. Bmal1 loss-of-function causes swollen mitochondria incapable of adapting to different nutrient conditions accompanied by diminished respiration and elevated oxidative stress. Consequently, liver-specific Bmal1 knockout (LBmal1KO) mice accumulate oxidative damage and develop hepatic insulin resistance. Restoration of hepatic Bmal1 activities in high-fat-fed mice improves metabolic outcomes, whereas expression of Fis1, a fission protein that promotes quality control, rescues morphological/metabolic defects of LBmal1KO mitochondria. Interestingly, Bmal1 homolog AHA-1 in C. elegans retains the ability to modulate oxidative metabolism and lifespan despite lacking circadian regulation. These results suggest clock genes are evolutionarily conserved energetics regulators.
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•Bmal1 controls rhythmic mitochondrial dynamics gene expression in the liver•The dynamic mitochondrial activity manages metabolic flexibility and oxidative stress•Bmal1 depletion causes enlarged, dysfunctional mitochondria and hepatic pathologies•C. elegans Bmal1 homolog AHA-1 regulates oxidative metabolism and extends lifespan
Mitochondrial dynamics plays an important role in metabolic adaptation to nutrient influx. Jacobi et al. reveal that the circadian regulator Bmal1 controls rhythmic mitochondrial dynamics gene expression in the liver. Hepatic Bmal1 gene deletion causes abnormal mitochondrial morphology, elevated oxidative damage, and metabolic inflexibility.
The nicotinamide adenine dinucleotide (NAD
/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) ...cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD
transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify
(
) as coessential with ETC components.
-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD
and NADH. Isolated mitochondria from cells lacking or overexpressing
have greatly decreased or increased NAD uptake in vitro, respectively. Moreover,
and
, a yeast mitochondrial NAD
transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.
Phosphatidylcholine (PC) is the major glycerophospholipid in eukaryotic cells and is an essential component in all cellular membranes. The biochemistry of de novo PC synthesis by the Kennedy pathway ...is well established, but less is known about the physiological functions of PC. We identified two unrelated patients with defects in the Kennedy pathway due to biallellic loss-of-function mutations in phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting enzyme in this pathway. The mutations lead to a marked reduction in PCYT1A expression and PC synthesis. The phenotypic consequences include some features, such as severe fatty liver and low HDL cholesterol levels, that are predicted by the results of previously reported liver-specific deletion of murine Pcyt1a . Both patients also had lipodystrophy, severe insulin resistance, and diabetes, providing evidence for an additional and essential role for PCYT1A-generated PC in the normal function of white adipose tissue and insulin action.
Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and ...metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism.
Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as ...proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.
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