Joel M. Goodman

The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology

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 endosomal–lysosomal 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.

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C‐terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.

An intimate collaboration between peroxisomes and lipid bodies

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.

The Gregarious Lipid Droplet

Cytoplasmic lipid droplets were considered until recently to be in the same category as glycogen granules, simple storage sites for energy, waxing and waning as metabolic energy needs dictated, but otherwise inert particles. It has become clear, however, that droplets are much more than isolated storage depots in the cell and that they can skate around on the cytoskeleton, physically interact with several organelles over short or long durations, and be beasts of burden, storing important molecules unrelated to lipids for later use. Although important clues to the panoply of droplet functions have come to light as a result of several proteomics studies, some behavioral qualities of this organelle were apparent from older morphological studies. Droplets are indeed gregarious. In this Minireview, after first considering the basic biochemical properties of lipid droplets, I shall focus on their interactions with other organelles, as manifest by morphological and dynamic studies and hinted at by proteomics. The important functions of droplets in storing and chaperoning proteins are well covered in a recent review (1) and will not be discussed here.

The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

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

The targeting and assembly of peroxisomal proteins: some old rules do not apply

Several proteins have been identified that catalyze the import of proteins into peroxisomes. Some recognize specific peroxisomal targeting sequences, but most probably work further downstream the import pathway. Recent evidence suggests that peroxisomal targeting and assembly do not follow the same rules as those for targeting and import into other organelles, such as the mitochondria and the endoplasmic reticulum, i.e. the import of unfolded proteins and subsequent folding within the organelle. Specifically, proteins may be translocated into the peroxisomal matrix in a folded or oligomerized state.

Demonstrated and inferred metabolism associated with cytosolic lipid droplets

Cytosolic lipid droplets were considered until recently to be rather inert particles of stored neutral lipid. Largely through proteomics is it now known that droplets are dynamic organelles and that they participate in several important metabolic reactions as well as trafficking and interorganellar communication. In this review, the role of droplets in metabolism in the yeast Saccharomyces cerevisiae, the fly Drosophila melanogaster, and several mammalian sources are discussed, particularly focusing on those reactions shared by these organisms. From proteomics and older work, it is clear that droplets are important for fatty acid and sterol biosynthesis, fatty acid activation, and lipolysis. However, many droplet-associated enzymes are predicted to span a membrane two or more times, which suggests either that droplet structure is more complex than the current model posits, or that there are tightly bound membranes, particularly derived from the endoplasmic reticulum, which account for the association of several of these proteins.

Proton ionophores prevent assembly of a peroxisomal protein

Peroxisomal matrix proteins are imported into the organelle posttranslationally. Here we report that proton ionophores disrupt the import and assembly of alcohol oxidase, a homo-octameric flavoprotein of the induced peroxisome from the methylotrophic yeast Candida boidinii. When drug is added to cells containing newly synthesized monomeric alcohol oxidase, octamerization fails to occur and a membrane-associated complex is formed instead. The formation of the complex, which appears to face the cytoplasmic side of the membrane, is reversed when drug is removed, leading to the generation of octamer. Surprisingly, when drug is added to cells containing newly assembled octamers, they dissociate into monomers. We suggest that both the complex and the labile octamer are intermediates in the normal assembly pathway of alcohol oxidase and that energy is required for import and maturation of this peroxisomal protein.

Seipin Is a Discrete Homooligomer

Seipin is a transmembrane protein that resides in the endoplasmic reticulum and concentrates at junctions between the ER and cytosolic lipid droplets. Mutations in the human seipin gene, including the missense mutation A212P, lead to congenital generalized lipodystrophy (CGL), characterized by the lack of normal adipose tissue and accumulation of fat in liver and muscles. In both yeast and CGL patient fibroblasts, seipin is required for normal lipid droplet morphology; in its absence droplets appear to bud abnormally from the ER. Here we report the first purification and physical characterization of seipin. Yeast seipin is in a large discrete protein complex. Affinity purification demonstrated that seipin is the main if not exclusive protein in the complex. Detergent sucrose gradients in H(2)O, and D(2)O and gel filtration were used to determine the size of the seipin complex and account for detergent binding. Both seipin-myc13 (seipin fused to 13 tandem copies of the myc epitope) expressed from the endogenous promoter and overexpressed seipin-mCherry form ∼500 kDa proteins consisting of about 9 copies of seipin. The yeast orthologue of the human A212P allele forms only smaller complexes and is unstable; we hypothesize that this accounts for its null phenotype in humans. Seipin appears as a toroid by negative staining electron microscopy. We speculate that seipin plays at least a structural role in organizing droplets or in communication between droplets and ER.

The lipid droplet—a well-connected organelle

Our knowledge of inter-organellar communication has grown exponentially in recent years. This review focuses on the interactions that cytoplasmic lipid droplets have with other organelles. Twenty-five years ago droplets were considered simply particles of coalesced fat. Ten years ago there were hints from proteomics studies that droplets might interact with other structures to share lipids and proteins. Now it is clear that the droplets interact with many if not most cellular structures to maintain cellular homeostasis and to buffer against insults such as starvation. The evidence for this statement, as well as probes to understand the nature and results of droplet interactions, are presented.