Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease.

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.

Essential role for diacylglycerol in protein transport from the yeast Golgi complex.

Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain. Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles.

Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis.

The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMG-CoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMG-CoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

The yeast BSD2-1 mutation influences both the requirement for phosphatidylinositol transfer protein function and derepression of phospholipid biosynthetic gene expression in yeast.

The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino- phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the "bypass Sec14p" phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidyl-inositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed.

The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity.

The Saccharomyces cerevisiae protein SEC14p is required for Golgi function and cell viability in vivo. This requirement is obviated by mutations that specifically inactivate the CDP-choline pathway for phosphatidylcholine biosynthesis. The biochemical basis for the in vivo relationship between SEC14p function and the CDP-choline pathway has remained obscure. We now report that SEC14p effects an in vivo depression of CDP-choline pathway activity by inhibiting choline-phosphate cytidylyltransferase (CCTase; EC 2.7.7.15), the rate-determining enzyme of the CDP-choline pathway. Moreover, this SEC14p-mediated inhibition of CCTase was recapitulated in vitro and was saturable. Finally, whereas the SEC14p-dependent inhibition of CCTase in vitro was markedly reduced under assay conditions that were expected to increase levels of phosphatidylinositol-bound SEC14p, assay conditions expected to increase levels of phosphatidylcholine-bound SEC14p resulted in significant potentiation of CCTase inhibition. The collective data suggest that the phosphatidylcholine-bound form of SEC14p effects an essential repression of CDP-choline pathway activity in Golgi membranes by inhibiting CCTase and that the phospholipid-binding/exchange activity of SEC14p represents a mechanism by which the regulatory activity of SEC14p is itself controlled.

Functional redundancy of CDP-ethanolamine and CDP-choline pathway enzymes in phospholipid biosynthesis: ethanolamine-dependent effects on steady-state membrane phospholipid composition in Saccharomyces cerevisiae.

It has been established that yeast membrane phospholipid content is responsive to the inositol and choline content of the growth medium. Alterations in the levels of transcription of phospholipid biosynthetic enzymes contribute significantly to this response. We now describe conditions under which ethanolamine can exert significant influence on yeast membrane phospholipid composition. We demonstrate that mutations which block a defined subset of the reactions required for the biosynthesis of phosphatidylcholine (PC) via the CDP-choline pathway cause ethanolamine-dependent effects on the steady-state levels of bulk PC in yeast membranes. Such an ethanolamine-dependent reduction in bulk membrane PC content was observed for both choline kinase (cki) and choline phosphotransferase (cpt1) mutants, but it was not observed for mutants defective in cholinephosphate cytidylyltransferase, the enzyme that catalyzes the penultimate reaction of the CDP-choline pathway for PC biosynthesis. Moreover, the ethanolamine effect observed for cki and cpt1 mutants was independent of the choline content of the growth medium. Finally, we found that haploid yeast strains defective in the activity of both the choline and ethanolamine phosphotransferases experienced an ethanolamine-insensitive reduction in steady-state PC content, an effect which was not observed in strains defective in either one of these activities alone. The collective data indicate that specific enzymes of the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis, while able to contribute to PC synthesis when yeast cells are grown under conditions of ethanolamine deprivation, do not do so when yeast cells are presented with this phospholipid headgroup precursor.

Purification and characterization of a late Golgi compartment from Saccharomyces cerevisiae.

We have devised a method for obtaining highly enriched membranes of a late yeast Golgi compartment, operationally defined by their containing the Kex2p protease, and generated four hybridoma cell lines that produced monoclonal antibodies directed against distinct Golgi membrane proteins (GMPs) (GMP36, GMP51, GMP77, and GMP95). Immunofluorescence and subcellular fractionation data indicated that, of the four GMPs analyzed, only GMP51 exhibited essentially an absolute colocalization with Kex2p. Also, as in the case of Kex2p, retention of GMP51 in yeast Golgi membranes was dependent on clathrin function. In contrast, the remaining three GMPs exhibited substantial, but not absolute, colocalization with Kex2p. The collective data are most consistent with a model where GMP36, GMP77, and GMP95 are present in all Kex2p-containing membranes, but Kex2p is present in only a subpopulation of membranes that contain these GMPs, thereby suggesting that either these particular GMPs exhibit overlapping distributions in compartments of the yeast Golgi complex or are also present in non-Golgi compartments. These findings are not consistent with the view that resident yeast Golgi proteins are generally restricted to a specific Golgi subcompartment, but they are consistent with the view that Golgi compartmental identity is determined by the relative mixtures of Golgi proteins that reside within individual cisternae.

A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes.

SEC14p is required for protein transport from the yeast Golgi complex. We describe a quantitative analysis of yeast bulk membrane and Golgi membrane phospholipid composition under conditions where Golgi secretory function has been uncoupled from its usual SEC14p requirement. The data demonstrate that SEC14p specifically functions to maintain a reduced phosphatidylcholine content in Golgi membranes and indicate that overproduction of SEC14p markedly reduces the apparent rate of phosphatidylcholine biosynthesis via the CDP-choline pathway in vivo. We suggest that SEC14p serves as a sensor of Golgi membrane phospholipid composition through which the activity of the CDP-choline pathway in Golgi membranes is regulated such that a phosphatidylcholine content that is compatible with the essential secretory function of these membranes is maintained.

SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast.

Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein.

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

The pathogenesis of peripheral aneurysms of the central nervous system: a subject review from the AFIP.

Most central nervous system aneurysms occur around the circle of Willis, and are congenital or arteriosclerotic in origin when in that location. Peripherally located aneurysms are either idiopathic or secondary to infection, tumor embolus (from choriocarcinoma and cardiac myxoma), Moyamoya disease, or trauma. The pathophysiologic features of these aneurysms are discussed.

Prognosis in meningioma through evaluation of skull bone patterns.

Sixty-three cases of meningioma were selected for study. Radiologically, they were divided into blastic and cystic groups. Tumors producing more blastic patterns infrequently recurred, unlike those that contained a lytic factor. The cases were also analyzed in regard to histologic type, but no relationship was found between histologic type and recurrence.