Lack of requirement for sterol carrier protein-2 in the intracellular trafficking of lysosomal cholesterol.

Previous work has established that the absence of peroxisomes, as occurs in Zellweger syndrome, is accompanied by the absence of cellular sterol carrier protein-2 (SCP2). In the present study, Zellweger-syndrome fibroblasts and peroxisome-deficient CHO-ZR78 cells were used to study the role of SCP2 in the intracellular transport of low density lipoprotein (LDL)-derived lysosomal cholesterol. By immunoblotting, peroxisome-deficient cells were confirmed to contain either no detectable SCP2 or far less SCP2 than corresponding normal cells. To monitor the transport of lysosomal cholesterol to the plasma membrane, we measured efflux of lysosomal cholesterol to HDL3 or phospholipid vesicles. SCP2-deficient cells, in comparison to normal cells, demonstrated little or no impairment in this efflux, suggesting that SCP2 is not required for the efficient delivery of lysosomal cholesterol to the plasma membrane. To examine the role of SCP2 in the delivery of lysosomal cholesterol to acyl-CoA:cholesterol acyltransferase (ACAT) in the rough endoplasmic reticulum (RER), the lysosomal and whole-cell cholesterol pools were differentially labeled, and then the ACAT-mediated esterification of each pool was measured in response to an 8-h incubation with native LDL. For both cholesterol pools, esterification was stimulated by LDL, and the responses in normal and Zellweger cells were similar, demonstrating that SCP2 is required for neither the stimulation of ACAT that follows LDL uptake nor for the transport of lysosomal cholesterol to the RER. These findings suggest that some major aspects of lysosomal cholesterol trafficking in cells can occur by mechanisms not involving SCP2.

Developmental regulation of sterol carrier protein 2 in chicken intestine and liver at time of hatching.

Sterol Carrier Protein 2, a protein thought to be involved in various aspects of intracellular sterol transport and metabolism, was studied in the small intestine and liver of chickens near hatching. In the intestine, a sudden shift in molecular mass, from 12 kDa to 64 kDa was noted by Western blotting on the day of hatching. Immediately post-hatching, the molecular mass returned to 12 kDa but the protein was several fold more abundant than prior to hatching. In the liver, several molecular mass forms were present at all ages examined but total shift to the 64 kDa form was not seen. Following hatching, however, the 12 kDa form became more abundant. Regardless of age of bird or molecular mass, the protein was found mainly in peroxisomes.

The presence and subcellular distribution of sterol carrier protein 2 in embryonic-chick tissues.

Transport of lipids from the yolk to the tissues of the chick embryo is slow during the first 2 weeks of development, but increases abruptly during the last week. Evidence suggests that the lipid traverses the cytoplasm of the yolk-sac membrane before secretion as lipoprotein into the fetal circulation. Little is known about the cytoplasmic transport of lipid in avian systems, but recently the presence of sterol carrier protein 2 (SCP2) was reported in chicken liver. Here we examine the cells of yolk-sac membrane, liver and small intestine for the presence of this protein as a function of the time of embryonic development. The quantity of SCP2 present in the embryonic cells did not appear to correlate with the rate of lipid flux in these tissues. The abrupt appearance of a high-molecular-mass form of SCP2 was detected in small intestine shortly before hatching, but the significance of this protein is not clear. The presence of SCP2 in these tissues was also confirmed by immunocytochemical techniques. Similarly to SCP2 of mammalian cells, avian SCP2 is localized in both peroxisome-like structures and mitochondria. To a lesser extent it is associated with the endoplasmic reticulum.

Purification, characterization and comparison with mammalian SCP2 of a chicken SCP2-like protein.

1. Three proteins have been isolated from chicken (Gallus domesticus) liver that bind antibodies directed against authentic rat sterol carrier protein2 (SCP2) and have similar molecular mass to the three major immunoreactive rat liver proteins (12 kDa, 30-36 kDa, 55-60 kDa). 2. Bile from both chicken and rat contains the high molecular mass immunoreactive species. 3. The chicken 12 kDa SCP2-like protein purifies similarly to rat SCP2 but the homogeneous chicken SCP2-like protein is dissimilar in amino acid composition and N-terminal amino acid sequence. 4. The activity of chicken SCP2-like protein differs from rat SCP2 in that it was consistent with fusion (transfer of both polar surface and non-polar core lipids) rather than transfer of polar lipids only.

Intracellular sterol trafficking.

Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.

Intracellular trafficking of sterols.

Cavalier-Smith (1981) has identified 22 characters that are universally present in eukaryotes but absent in prokaryotes. Of these, he argues that one, exocytosis, might have been the driving force behind the evolution of modern eukaryotic cells. Bloom and Mouritsen (1988) further argue that sterols may have removed an evolutionary bottleneck to cytosis. Therefore, the advent of sterols in membranes might have been the single feature that led to eukaryote evolution. The evolutionary advantage conferred by cholesterol is associated primarily with plasma membrane function, since the majority of cellular free cholesterol resides in that membrane. However, sterol synthesis occurs in the ER; therefore, the cell must have a mechanism for transporting sterol to the plasma membrane and its regulation. As has been pointed out in this review, much remains to be elucidated in the study of intracellular sterol trafficking. To date, neither diffusion nor vesicle-mediated transport can be fully confirmed or ruled out. Microtubule and microfilament involvement appears important in some routes (e.g., mitochondria) but not in others. In addition, trafficking roles of cytoplasmic lipoproteinlike particles have not been addressed. Finally, although some "sterol carrier proteins" demonstrate the ability to mediate intervesicular transfer of cholesterol in vitro, the true physiological role of these proteins remains obscure. Future research in this field awaits the refinement of available techniques. Particularly valuable would be cytochemical methods for detection of sterol at the ultrastructural level. Possibly, direct microscopic visualization of radiolabeled components in cells represents the necessary approach. Purification of elements carrying newly synthesized sterols would allow the proteins mediating transport to be identified. Continued analysis of mutants defective in transport, such as in type C Niemann-Pick disease, will shed light on this complex problem. The importance of extracellular trafficking of cholesterol owing to its involvement in the progression of atherosclerosis, has been emphasized in recent years. Little emphasis has been placed on intracellular trafficking of sterol; however, it can be argued that such transport also plays a major role in atherosclerosis, possibly by fueling retrotransport of cholesterol to the liver and secretion in the bile. Therefore, we hope this review will serve to stimulate research interest in this area.

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver.

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.

A single tRNA (guanine)-methyltransferase from Tetrahymena with both mono- and di-methylating activity.

A tRNA (guanine-2) methyltransferase has been purified to homogeneity from the protozoan Tetrahymena pyriformis. The enzyme methylates purified E. coli tRNAs which have a guanine residue at position 26 from the 5' end; it also methylates tRNA prepared from the m22G- yeast mutant trm 1. This methyltransferase is therefore equivalent to the guanine methyltransferase 2mGII found in mammalian extracts. The purified 2mGII from Tetrahymena is capable of forming both N2-methylguanine and N22-dimethylguanine on a single tRNA isoaccepting species; under conditions of limiting tRNA or long reaction times the predominant product is dimethylguanine. Analysis of the products formed under varying reaction conditions suggests that dimethylguanine formation is a two step process requiring dissociation of the enzyme-monomethylated tRNA intermediate.