Lipodystrophy is a disorder characterized by a loss of adipose tissue often accompanied by severe hypertriglyceridemia, insulin resistance, diabetes, and fatty liver. It can be inherited or acquired. The most severe inherited form is Berardinelli-Seip Congenital Lipodystrophy Type 2, associated with mutations in the BSCL2 gene. BSCL2 encodes seipin, the function of which has been entirely unknown. We now report the identification of yeast BSCL2/seipin through a screen to detect genes important for lipid droplet morphology. The absence of yeast seipin results in irregular lipid droplets often clustered alongside proliferated endoplasmic reticulum (ER); giant lipid droplets are also seen. Many small irregular lipid droplets are also apparent in fibroblasts from a BSCL2 patient. Human seipin can functionally replace yeast seipin, but a missense mutation in human seipin that causes lipodystrophy, or corresponding mutations in the yeast gene, render them unable to complement. Yeast seipin is localized in the ER, where it forms puncta. Almost all lipid droplets appear to be on the ER, and seipin is found at these junctions. Therefore, we hypothesize that seipin is important for droplet maintenance and perhaps assembly. In addition to detecting seipin, the screen identified 58 other genes whose deletions cause aberrant lipid droplets, including 2 genes encoding proteins known to activate lipin, a lipodystrophy locus in mice, and 16 other genes that are involved in endosomallysosomal trafficking. The genes identified in our screen should be of value in understanding the pathway of lipid droplet biogenesis and maintenance and the cause of some lipodystrophies. BSCL2 ͉ lipid bodies

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5-(␤,␥-imino)triphosphate and adenosine 5-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl 2 , substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction. ATP-binding cassette (ABC)1 transporters are ubiquitous membrane proteins that couple ATP hydrolysis to the energydependent transport of a wide variety of molecules across lipid bilayers. They comprise the single largest gene family in several sequenced prokaryotic genomes (1). Mutations in human ABC transporters underlie diseases such as cystic fibrosis, hypercholesterolemia, adrenoleukodystrophy, and Stargardt's disease, while multidrug resistance in cancer cells and infectious microorganisms often arises from the overexpression of ABC transporters that serve as drug efflux pumps (2, 3).The ABC transporters share an invariant domain organization of two conserved cytoplasmic nucleotide binding cassettes associated with two transmembrane (TM) domains (2). The cassettes contain three highly conserved motifs required for nucleotide binding and hydrolysis: the Walker A site (GX 4 GK(S/T), where X ϭ any residue) and the Walker B site (RX 6 -8 ⌽ 4 D, where ⌽ ϭ hydrophobic residue) (4), which reside in the ␣␤ core of the cassette (5-11), and the LSGGQ signature sequence (1, 2), which lies more toward the periphery of the cassette in an ␣-helical subdomain (5-11). The TM domains that mediate the movement of the structurally diverse solutes exhibit less sequence conservation (1, 2). The organization of prokaryotic transporter operons and of single polypeptide chain transporters suggests that the minimal functional unit consists of at least two cassettes and two TM domains (5).The crystal structures of a homodimeric half...

Pah1p promotes lipid droplet assembly independent of its role in triacylglycerol synthesis.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid–cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid β oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies (“gnarls”), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Loss-of-function mutations in seipin cause severe lipodystrophy, yet seipin's function in incompletely understood. Seipin is shown here to be important specifically for initiation of droplet formation, and a deletion mutant allows dissection of this function from maintenance of droplet morphology and vectorial droplet budding.