Thomas W. Keenan

Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases

Abstract We report the human DNA and protein sequence of adipophilin and its association with the surface of lipid droplets. The amino acid sequence of human adipophilin has been determined by using cDNA clones from several tissues and confirmed by the reverse transcription/polymerase chain reaction method and Edman sequencing. The open reading frame of adipophilin encodes a polypeptide with a calculated molecular weight of 48.1 kDa and an isoelectric point of 6.72. By immunofluorescence and electron-microscopic localization with newly raised specific poly- and monoclonal antibodies, we show that this protein is not restricted to adipocytes as previously indicated by studies of the mouse homologous protein, adipose-differentiation-related protein. Adipophilin occurs in a wide range of cultured cell lines, including fibroblasts and endothelial and epithelial cells. In tissues, however, expression of adipophilin is restricted to certain cell types, such as lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and steatosis or fatty change hepatocytes in alcoholic liver cirrhosis. Our results reveal adipophilin as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells.

Localization of glycosyl transferase activities in a Golgi apparatus-rich fraction isolated from rat liver.

Abstract A Golgi apparatus-rich fraction isolated from rat liver catalyzed the transfer of galactose from UDP-galactose to N-acetylglucosamine with the formation of N-acetylaminolactose as well as the transfer of glucosamine from UDP-N-acetyl- glucosamine to endogenous protein acceptors. Based on enzymatic and morphological criteria, Golgi apparatus fractions were estimated to contain at least 80% Golgi apparatus-derived material. Approximately half of the total glycosyl transferase activities of the original homogenates was recovered in the Golgi apparatus fraction. The glycosyl transferase activities of purified endoplasmic reticulum fractions were much lower than those of the Golgi apparatus. Plasma membrane fractions as well as the soluble supernatant fraction contained little or no activity.

Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation

Cells of the cyanobacterium Nostoc commune secrete a complex, high molecular weight, extracellular polysaccharide (EPS) which accumulates to more than 60% of the dry weight of colonies. The EPS was purified from the clonal isolate N. commune DRH1. The midpoint of the membrane phase transition (Tm) of desiccated cells of N. commune CHEN was low (Tmdry = 8 °C) and was comparable to the Tm of rehydrated cells((Tm)H20 = 6 °C). The EPS was not responsible for the depression of Tm. However, the EPS, at low concentrations, inhibited specifically the fusion of phosphatidylcholine membrane vesicles when they were dried in vitro at0% relative humidity (−400 MPa). Low concentrations of a trehalose:sucrose mixture, in a molar ratio which corresponded with that present in cells in vivo, together with small amounts of the EPS, were efficient in preventing leakage of carboxyfloroscein (CF) from membrane vesicles. Freeze-fracture electron microscopy resolved complex changes in the structure of the EPS and the outer membrane in response to rehydration of desiccated cells. The capacity of the EPS to prevent membrane fusion, the maintenance of a low Tmdry in desiccated cells, and the changes in rheological properties of the EPS in response to water availability, constitute what are likely important mechanisms for desiccation tolerance in this cyanobacterium.

Butyrophilin, an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of Mr 12000-16000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.

Historical Perspective: Milk Lipid Globules and Their Surrounding Membrane: A Brief History and Perspectives for Future Research

Most of the lipids in milk are triacylglycerols that occur in globules surrounded by a membrane derived from cellular membranes. This membrane, the milk-fat or milk-lipid globule membrane (MLGM),2 surrounds globules during the process of their secretion from the cell. The nature and cellular origin of the milk lipid globule membrane has been the subject of a considerable amount of research. Milk lipid globules originate as very small lipid droplets formed on or in the endoplasmic reticulum followed by release into the cytosol. These droplets consist of a triacylglycerol-rich core coated with a layer of proteins and polar lipids. How these droplets are formed, how they can grow in volume, how they move through the cell, and how they are secreted are questions that have been the basis for a number of investigations. While the general outlines of droplet formation, growth, movement, and secretion are known, virtually no molecular details of any of these processes have been elucidated. In this article I have presented a brief historical account of research on milk fat globules, their surrounding membrane, and on aspects of the intracellular origin, growth, and secretion of milk lipid globules. I have also attempted to call attention to those areas where further research is needed to gain a better understanding of the processes involved.

Ceramide excluded from cell-free vesicular lipid transfer from endoplasmic reticulum to Golgi apparatus. Evidence for lipid sorting

The distribution and cell-free transfer of ceramide and other lipids were compared using highly purified fractions of endoplasmic reticulum, transitional endoplasmic reticulum, transition vesicles and Golgi apparatus from rat liver. Ceramides were present in both endoplasmic reticulum and Golgi apparatus where they represented between 0.3 and 1% of the total lipids. Ceramides, however, were much reduced or absent (< 0.05%) from transition vesicles. Transition vesicles were induced to form from transitional endoplasmic reticulum by incubation with ATP and a cytosol fraction. When transfer of [14C]choline-labeled phosphatidylcholine from transitional endoplasmic reticulum to Golgi apparatus was followed, transition vesicles were more efficient in transfer than the transitional endoplasmic reticulum from which they were derived. This transfer was temperature- and ATP-dependent and inhibited by N-ethylmaleimide. When transfer of [3H]ceramide was followed, there was little or no transfer via transition vesicles and that transfer which occurred was temperature-, ATP- and N-ethylmaleimide independent. Transfer of ceramide in the cell-free system did occur from endoplasmic reticulum to Golgi apparatus but via a non-vesicular mechanism that was temperature-dependent but not dependent on ATP or cytosol, alone, or in combination, nor was it inhibited by N-ethylmaleimide. A component of phosphatidylcholine transfer exhibited similar characteristics. The results provide evidence for two distinct mechanisms for cell-free transfer of lipids from endoplasmic reticulum to Golgi apparatus. The first is via 50 to 70 nm transition vesicles which is temperature- and ATP-dependent, inhibited by N-ethylmaleimide and from which ceramides are excluded. The second is non-vesicular, temperature-dependent, and neither ATP- nor cytosol-dependent. It accounts for the bulk of the ceramide transfer. As a result during cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus, lipid sorting occurs such that ceramides are largely absent from the transition vesicles and, apparently are delivered to the Golgi apparatus by another mechanism.

Butyrophilin and xanthine oxidase occur in constant molar proportions in milk lipid globule membrane but vary in amount with breed and stage of lactation

SummaryButyrophilin and xanthine oxidase, major proteins of milk lipid globule membrane, both accounted for significantly higher percentages of total protein in membrane samples from Holstein than from Jersey animals. Both were high in membranes from animals in early lactation, both decreased in amount as lactation progressed to the midpoint, and then both rose in amount toward the end of lactation. In samples from both Holstein and Jersey animals, butyrophilin and xanthine oxidase were present in constant molar proportions of about 4∶1. These proteins co-enriched together with low molecular weight GTP-binding proteins in a high salt and nonionic detergent insoluble fraction of milk lipid globule membrane. Butyrophilin and xanthine oxidase content of membranes was not related to milk lipid globule diameter, suggesting that these proteins alone may not be involved solely in anchoring the membrane to the lipid globule surface. However, the possibility that a complex composed in part of butyrophilin and xanthine oxidase serves an anchoring function remains a possibility.

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.

Origin of Milk Fat Globules and the Nature of the Milk Fat Globule Membrane

Available evidence suggests that milk fat globules originate from endoplasmic reticulum of mammary epithelial cells. The actual process by which these globules are formed is unknown, but there are indications that triglyceride-containing vesicles which bleb from endoplasmic reticulum may serve as nucleation sites for globules. After formation, lipid droplets grow within the cell and this growth appears to be mediated by fusion of triglyceride-containing vesicles with intracellular lipid droplets. After formation, lipid droplets migrate to apical regions of cells, from where they are secreted into the alveolar lumen. Some evidence that microtubules and microfilaments may be involved in the intracellular migration of lipid droplets has been obtained, but this evidence is equivocal. Lipid droplets are released from the cell by envelopment in specialized regions of apical plasma membrane. Membranes of secretory vesicles may also make a contribution to the membrane which envelops lipid droplets. As reviewed herein, the membrane which surrounds fat globules in milk has been extensively characterized.

Rehydration induces rapid onset of lipid biosynthesis in desiccated Nostoc commune (Cyanobacteria)

Water, which contained [1,3-3H]glycerol, [35S]sodium sulfate, or [32P]sodium orthophosphate, was used to rehydrate air-dried cells of the desiccation-tolerant filamentous cyanobacterium Nostoc commune. The cells retained their capacities for the uptake and transport of all three compounds and, in response to rewetting, they mobilized the radiolabels into lipid precursors and initiated complex lipid biosynthesis. The onset of these events, measured in short-term, long-term and pulse-chase labeling experiments, was judged to be very rapid. The radiolabeled pool sizes of the major membrane species phosphatidylglycerol (PG) and sulfoquinovosyl diacylglycerol (SQDG) reached steady-state within several minutes, while those of the two abundant membrane glycolipids, mono- and di-glycosyldiacylglycerol (MGDG, DGDG), achieved uniform labeling within 2 h. The pattern of sulfolipid synthesis was generally more complex than the other lipid species. Analysis of the maturation of SQDG through differential labeling provided the only example of a lag in lipid maturation during the early stages (minutes) of cell rehydration. In this instance, the lag appeared to be associated specifically with the incorporation of 35SO3- by the sulfoquinovose. During the initial 2 h of rewetting there was complete turnover of 3H-label in the pools of the principal lipid precursors 1,2-sn-diacylglycerol and 1,3-diacylglycerol. In contrast, the accumulation of label by the major lipid of the heterocyst cell-wall, a non-saponifiable glycolipid, became detectable only after 24 h of rewetting. The present data are discussed in relation to the basis for desiccation tolerance in N. Commune.