This article is available online at http://www.jlr.org developed very early during evolution. Cytosolic lipid droplets (LDs) are the main reservoir of lipids and are common to many if not all eukaryotic cells ( 1 ). LDs have gained much recent interest because of their regulatory role in lipid homeostasis and their implication in metabolic diseases such as obesity and type 2 diabetes ( 2-4 ). They have a unique structure composed of a hydrophobic core surrounded by a phospholipid monolayer containing a specifi c protein composition. Perilipin family proteins and lipid metabolizing enzymes are the most abundant proteins ( 5, 6 ), and proteomic studies have identifi ed many additional constituents ( 7 ). Little is known how proteins are specifi cally targeted to lipid droplets ( 8, 9 ). The biogenesis of LDs likely involves the endoplasmic reticulum, but the mechanism has not been solved and is debated intensely ( 2,3,10 ).Triglycerides are the main species of neutral lipids stored within the LDs of most cell types. The predominant biosynthetic pathway requires three activated fatty acids for each triglyceride molecule. The fatty acids are taken up from the extracellular medium or are derived from endogenous metabolism by fatty acid synthase. In fact, addition of fatty acids is a very effi cient way to induce the formation of LDs ( 4 ). However, fatty acids are chemically quite inert and need to be activated by esterifi cation with CoA ( 11 ). This activation is catalyzed by the family of acylCoA synthetases ( 12 ); physiologically highly relevant are the long chain (ACSL1, -3, -4, -5, -6) and very long chain (ACSVL1, -2, -3, -4, -5, and -6) fatty acyl-CoA synthetase subfamilies ( 13 ). Apart from their obvious enzymatic role, additional functions have been suggested for ACS(V)L family proteins: metabolic channeling of fatty acids toward specifi c metabolic fates [e.g., phospholipid synthesis vs.Abstract Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leafl et of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced b...

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Pseudoenzymes have emerged as key regulatory elements in all kingdoms of life despite being catalytically nonactive. Yet many factors defining why one protein is active while its homologue is inactive remain uncertain. For pseudoenzyme−enzyme pairs, the similarity of both subunits can often hinder conventional characterization approaches. In plants, a pseudoenzyme, PDX1.2, positively regulates vitamin B 6 production by association with its active catalytic homologues such as PDX1.3 through an unknown assembly mechanism. Here we used an integrative experimental approach to learn that such pseudoenzyme−enzyme pair associations result in heterocomplexes of variable stoichiometry, which are unexpectedly tunable. We also present the atomic structure of the PDX1.2 pseudoenzyme as well as the population averaged PDX1.2−PDX1.3 pseudoenzyme− enzyme pair. Finally, we dissected hetero-dodecamers of each stoichiometry to understand the arrangement of monomers in the heterocomplexes and identified symmetry-imposed preferences in PDX1.2−PDX1.3 interactions. Our results provide a new model of pseudoenzyme−enzyme interactions and their native heterogeneity.

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.