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6732-6738. 8 Baudier, J., Gtasser, N. and G6rard, D. (1986)

J. Biol. Chem. 261, 8192-8203 9 Dell'Angelica, E. C., Schleicher, C. H. and

Santome, J. A. (1994) J. Biol. Chem. 269, 28929-28936

10 F6hr, U. G. et al. (1995) J. Biol. Chem. 270, 21056-21061

11 Zimmer, D. B., Cornwall, E. H., Landar, A. and Song, W. (1995) Brain Res. Bull. 37, 417-429

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13 Reeves, R. H. et al. (1994) Proc. Natl Acacl. Sci. USA 91, 5359-5363

14 Lau, W., Devery, J. M. and Geczy, C. L. (1995) J. Clin. Invest. 95, 1957-1965

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18 Donato, R. (1991) Cell Calcium 12, 713-726

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297-303

The biology of HMG-CoA reductase: the pros of

contra-regulation

Randolph Hampton, Dago Dimster-Denk and Jasper Rine

HydroxymethylglutaryI-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived in- cluding cholesterol and prenyl moieties. The regulation of HMG-R is com- plex and includes feedback control, cross-regulation by independent bio- chemical processes and contra-reguiation of separate isozymes. From studies in yeast, these separate modes of regulation can be considered in an integrated fashion.

HMGICoA REDUCTASE (HMG-R), by catalyzing the synthesis of mevalonic acid from HMG-CoA, plays a critical role in the production of the large family of molecules derived from mevalonic acid by the mevalonate pathway (Fig. 1). Although the mevalonate pathway is best known as the source of sterols such as cholesterol ] , products of the mevalonate pathway comprise a stagger- ing array of molecules that vary among species, cell type and physiological

R. Hampton is at the Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; D. Dimster-Denk is at the Division of Biology, HHMI, California Institute of Technology, Pasadena, CA 91125, USA; and J. Rine is at the Division of Genetics, MCB Dept, 401 Barker Hall, University of California at Berkeley, Berkeley, CA 94720, USA.

140

state 2-4. Saccharomyces cerevisiae has been used as a model for HMG-R syn- thesis and has revealed numerous per- plexing features of HMG-R structure and regulation. In this review, we will dis- cuss the HMG-R protein family, includ- ing the structural themes and the regu- latory modes encountered in diverse organisms. We will then focus on HMG-R in yeast, where most of the features of HMG-R biology are found and where they can be viewed in an integrated fashion.

The HMG.R protein family HMG-R is present in archaebacteria 5

and presumably in all eukaryotes ~-]~ A more distant relative of HMG-R has also been described in eubacteria u. The members of the HMG-R protein family consist of an extremely conserved cata- lytic domain and a highly diversified

�9 1996, Elsevier Science Ltd

membrane-anchoring region (Fig. 2). All versions of the protein have a highly conserved carboxy-terminal domain that is responsible for the conversion of HMG-CoA to mevalonic acid. Examples of the extent of conservation of cata- lytic function include the human en- zyme functioning in yeast 12 and the ar- chaeal enzyme being potently inhibited by lovastatin, a clinically used competi- tive inhibitor of mammalian HMG-R 5.

The individual catalytic domains of HMG-R are fused to a bewildering array of amino-terminal regions (Fig. 2). In most cases these amino-terminal re- gions appear to contain membrane- spanning sequences that anchor the protein in the endoplasmic reticulum (ER), but the archaeal enzyme is fused to a hydrophilic 21-residue peptide 5. The primary sequence and the result- ant structures of these amino termini are quite different. Those in plants have two putative transmembrane spans 2,1~ the metazoan HMG-Rs all have eight transmembrane spans 6;,]3, whereas yeast HMG-R appears to have seven transmembrane spans ]2'14. Furthermore, although comparisons among members of the same phylogenetic kingdom (e.g. tomato vs potato, or hamster vs sea urchin) often reveal primary sequence similarity in the amino-terminal regions, comparisons between the amino ter- mini of different kingdoms reveal no similarity at all. Given that the catalytic domain can function without membrane attachment, both in the natural setting of the archaeal HMG-R s and in engi- neered forms of other HMGoR pro- teins 15,16, why do so many HMG-Rs ap- pear to be attached to a membrane?

The HMG-R family also displays an- other level of complexity. In many in- stances, including all plants examined 4J7, S. cerevisiae ~2 and Dictyostelium (A. De Lozonne, pers. commun.), there are

PIi: S0968-0004(96) 10017-7

TIBS 21 - APRIL 1996 REVIEWS multiple isozymes of HMG-R within a given organism. It appears that meta- zoans have only a single enzyme. The dif- ferent isozymes within an organism are homologous and, thus, probably arose by duplication followed by significant sequence divergence. In comparing the HMG-R paralogues of a given organism, it is clear that the transmembrane re- gions display greater divergence than the catalytic domains ]~ Perhaps these individual transmembrane regions im- part distinct regulatory, targeting or functional properties to relatively simi- lar enzymatic activities.

The regulation of HMG-R The regulation of HMG-R, in the

broadest sense, fails into two cat- egories: feedback inhibition, whereby products from the mevalonate pathway regulate the amount of HMG-R activity by a variety of mechanisms; and cross- regulation, in which disparate biochemi- cal processes modulate levels of HMG-R activity.

Feedback inhibition. HMG-R activity is re- duced in response to products from the mevalonate pathway. Several other pro- teins involved in sterol metabolism are similarly regulated, including HMG-CoA synthase and the low-density lipopro- tein (LDL) receptor 1. Most studies of HMG-R feedback control have used mam- malian tissues and cells because of the medical importance of controlling sterol synthesis. When products of the path- way are low, the activity of HMG-R is high. When products of the pathway are abundant, the activity is low. In mam- mals, feedback regulation is achieved by coordinate modulation of the syn- thesis and degradation of the protein. For example, when pathway flux is low- ered, both transcription and translation of HMG-R are increased and the degra- dation rate is slowed, bringing about a coordinated increase in the steady-state level of the enzyme. A key issue is whether transcription, translation and stability of the HMG-R molecule all re- spond to the level of a common signal by the same sensing mechanism, or whether multiple, independent signal processes all occur simultaneously.

Studies on the mammalian enzyme have revealed that the amino-terminal membrane anchor is both necessary and sufficient to mediate regulated degra- dation of HMG-R ~s,18, which occurs in the ER itself along with the degradation of a growing list of other proteins 19.

Cross-regulation, The activity of HMG-R is also regulated by processes that are

HMG-CoA

3AcCoA

Early - I

HMG-R < "

mevalonate

thousands of products

oxygen independent

Late

> squalene > sterols

Figure 1 The simplified mevalonate pathway divided into early and late portions. HMG-CoA reduc- tase (HMG-R) catalyzes the indicated reaction in the early, oxygen-independent portion. Squalene is the product that defines the boundary with the late, oxygen-dependent portion of the pathway, as the next reaction (squalene epoxidation) is strictly dependent on oxy- gen. Several subsequent steps in the synthesis of sterols are also dependent on oxygen as well (not shown).

independent of the mevalonate path- way. These modes of control work inde- pendent of feedback regulation and are referred to as cross-regulation of HMG-R. The large number of biologi- cal processes that require distinct mevalonate pathway products implies that there could be many independent modes of cross-regulation.

The activity of the HMG-R catalytic domain can be down-modulated by phosphorylation with AMP-dependent kinase 2~ The phosphorylation of the HMG-R has no discernible effects on

stability 22, but rather alters the kinetic parameters of the enzyme. HMG-R phosphorylation is hypothesized to co- ordinate sterol synthesis with cellular energy charge 2~.

Another striking example of HMG-R cross-regulation in mammals occurs dur- ing stresses related to the invasion of pathogens. The potent bacterial toxin LPS, or certain cytokines that are pro- duced in response to LPS, cause a dras- tic increase in the levels of HMG-R mRNA in the liver and concomitant increases in the enzyme activity 23,24. The increase

(a) Archaeal ( ~ (b) Plant

d more

(d) Mammals ( ~

Figure 2 A stylized depiction of the basic structures of the large subgroups of the HMG-CoA reduc- tases (HMG-Rs) mentioned in the text. The same color means that there is similar size and hydrophobicity plot, and some level of detectable sequence similarity. The red portions represent the highly conserved, functionally analogous carboxy-terminal catalytic domains. The various colored amino-terminal regions represent the broad variations in these domains between the different groups. Multiple isozymes are indicated by multiple depictions of a given schematic.

1.41

tEVIEWS . . . . . . . . . . . . . . . TIBS 21 -APRIL 1996

in HMG-R mRNA is not a result of feed- back regulation from the mevalonate pathway, because other mRNAs that are similarly regulated by feedback control (e.g. LDL-receptor mRNA) remain unaf- fected. Rather, it appears that the physi- ological signals that herald an infection can rapidly and specifically induce HMG-R mRNA synthesis, perhaps through the same stress-related signaling path- ways that LPS and cytokines activate 2s. The lack of parallel derepression of other mevalonate-pathway enzymes might mean that the induction of HMG-R by pathogen stress has some purpose distinct from the need for increased mevalonate-pathway products.

In plants, cross-control of HMG-R is more intricate owing to the presence of multiple isozymes. Again, injury or pathogen invasion often triggers the in- duction of some HMC~R isozymes and the repression of others 26. Thus, the role of HMG-R as a pathogen-stimulated stress protein appears quite general in biology. In the case of plants, many of the molecules used to fight injury and invasion are from distinct branches of the mevalonate pathway. Perhaps the isozyme induced by an invading organ- ism can specifically promote produc- tion of a required mevalonate~erived pathocidal agent. One problem with this model is how mevalonate, a soluble product of HMG-R, could be directed to a specific, late branch of the pathway

through being synthesized by a specific isozyme of HMG-R. The principle of metabolic channeling has been sug- gested in this context and deserves fur- ther investigation 4.

The HMC~R isozymes of plants are also controlled developmentally. In the tom- ato, different isozymes are synthesized in particular tissues 26 (S. Jenkins and W. Gruissem, pers. commun.). Why such similar catalytic domains are differen- tially synthesized is not clear. Perhaps the specific isozymes used in particular tissues reflect distinct functions that might include targeting to a specific cel- lular location, regulated stability, ability to alter membrane structure or affinity for a particular multi-enzyme complex. It is also possible that the subtle differ- ences between the catalytic domains can direct flux along branches of the pathway that have distinct roles in dis- tinct tissues.

HMG-R in yeast Yeast has two isozymes of I-IMG-R

and both display feedback regulation as well as cross-regulation by oxygen. Many of the separate features of HMG-R biology described in plants or meta- zoans are observable in this genetically tractable organism, allowing an inte- grated view of the interplay of these separate features.

The isozymes of yeast HMG-R are called Hmglp and Hmg2p, and are en-

Hmglp early pioducts ~

Hmglp

T early products

late products I I I

Hmg2p

anaerobic

Figure 3 A model for the integrated action of oxygen (cross-) regulation and mevalonate product (feedback) regulation on the yeast HMG-CoA reductases (HMG-R) isozymes. Oxygen- and mevalonate-pathway products control the synthesis of each isozyme, both by repression (red bars) and stimulation (green arrows). The degradation of Hmg2p is also controlled by feedback regulation (blue)~ The feedback regulation of Hmg2p degradation occurs by early products stimulating the degradation. Oxygen is also required for the synthesis of late products from squalene, and that dependency is also depicted by a green arrow.

coded by the HMG1 and HMG2 genes, respectively ~2 . The catalytic domains of the two isozymes are extremely similar to each other (93% identical) and are significantly homologous to catalytic re- gions from other organisms. By con- trast, the hydrophobic amino termini are about 50% identical to each other but have no sequence or structural similarity to any other HMG-R amino termini 12-~4.

Feedback regulation of HMG-R in yeast Both yeast isozymes display feed-

back regulation: drugs or genetic per- turbations that lower flux through the mevalonate pathway result in increased steady-state levels of HMG-R. However, examination of the mechanism(s) of this feedback regulation has revealed striking differences between the two isozymes16, 27.

The feedback regulation of Hmglp appears to occur entirely at the level of mRNA translation 27. The range of regu- lation of Hmglp translation by feedback control is similar in magnitude to that observed in mammalian cells 28,29. The signals from the mevalonate pathway that control the translation rate of Hmglp are derived from early pathway products, before the production of squalene (Figs 1 and 3), as are the corresponding signals in mammalian cells 2s. The feedback control of Hmg2p synthesis has not been studied as com- pletely. It appears that the production of Hmg2p, by contrast with Hmglp, is modulated by molecules synthesized late in the pathway, either after, or at the level of squalene (D. Dimster-Denk and J. Pine, unpublished).

The degradation of one yeast HMG-R isozyme, Hmg2p, is regulated by the mevalonate pathway in the yeast cell: high pathway flux causes a high degra- dation rate and low pathway flux causes a slow degradation rate ~6. The mevalonate-derived signals for regu- lated degradation of Hmg2p lie early in the pathway, upstream of squalene. An early product of the pathway is also a critical determinant of HMG-R stability in mammalian cells 3~ and in such cells, the degradation of Hmg2p occurs in the ER. By contrast, the Hmglp isozyme is extremely stable.

The non-catalytic amino terminus of yeast Hmg2p is necessary and sufficient for regulated degradation of the Hmg2p protein. Thus, the amino termini of Hmg2p and human HMG-R are function- ally analogous, yet lack any sequence similarity. That the two isozymes of

142

TIBS 21 - APRIL 1996 REVIEWS HMG-R in yeast display such distinct degradative behaviors (Hmglp-stable, Hmg2p-regulated degradation) provides an opportunity to understand the se- quence determinants of degradation of ER-membrane-bound proteins, and how that process is regulated. Furthermore, the properties of Hmglp and Hmg2p re- inforce the idea that the amino termi- nus of an HMG-R isozyme can indeed impart distinct regulatory functions to that protein.

Why do the two enzymes display such different modes of feedback regu- lation of both synthesis and degradation when either is sufficient for life and they are usually coexpressed? By con- sidering the cross-regulation of HMG-R along with the feedback regulation some possible explanations emerge.

Cross-regulation of HMG-R by oxygen The expression of both isozymes of

HMG-R in yeast is regulated by oxy- gen 31,32 and can be described as 'contra- regulation', in which two isozymes are regulated in opposite fashion by the same stimulus. There are numerous other examples of contra-regulated pro- teins in yeast, including the Cox5p isozymes 33 and in some circumstances, the two Ras proteins 34. Because many yeast proteins are synthesized as pairs of isozymes derived from duplicated genes, it is entirely possible that contra- regulation plays a broad role in the con- trol of yeast gene expression.

The level of heme provides the molecular signal that causes contra- regulation of the HMG-R isozymes. Heine requires oxygen for its synthesis and is widely used as a gauge of oxygen availability 35. Studies of heme-regulated HMG-R expression 3~,32 have led us to the following model: when oxygen tension is high, as in aerobic growth, the pro- portion of Hmglp in the cell is high and that of Hmg2p is low. When oxygen ten- sion is low, as in semi- or purely an- aerobic conditions, the proportion of Hmglp is low and that of Hmg2p is high. Heme levels control the transcrip- tion of HMGI and HMG2 by two sep- arate mechanisms. Heme-stimulated transcription of the HMG1 gene is medi- ated by the Haplp transcriptional acti- vator, as are numerous other oxygen- stimulated genes 32,35. The mechanistic details of the aerobic repression and low-oxygen stimulation of HMG2 tran- scription are not yet clear. The oxygen- dependent transcriptional repressor Roxlp appears to have no role in the re- pression of the HMG2 gene, in spite of

the presence of a perfect Roxlp-binding consensus sequence in the HMG2 pro- moter (13. Dimster-Denk and J. Rine, unpublished).

The net effect of these complex ef- fects of oxygen on HMG-R is easily de- scribed: in aerobiosis, the predominant isoform is Hmglp. Upon switching to anaerobiosis, the predominant isoform switches from Hmglp to Hmg2p. Again, when considered in isolation the elabo- rate machinations that bring about this switch are hard to understand. Why does the cell replace one isoform with another one that has a catalytic domain of nearly identical sequence and activ- ity? lmmunolocalization experiments on each isozyme indicate that the distri- butions of each protein are similar (A. Koning and R. Wright, pers. com- mun.), suggesting that differences in isozyme synthesis do not reflect a re- quirement for HMG-R localization to dif- ferent parts of the cell during lowered oxygen availability. So why go to all of this trouble just to end up with a nearly identical catalytic activity with similar subcellular distribution?

The mevalonate pathway comes up for air The perplexing features that arise

when the feedback regulation or the oxygen regulation of yeast HMG-R are considered separately become easier to understand when both axes of regu- lation are considered together. Oxygen plays a critical role in the late meval- onate pathway and absolutely no role in the early mevalonate pathway (Fig. 1). Oxygen is required for the epoxidation of squalene to squalene epoxide, which is then cyclized to lanosterol, in addi- tion to other steps in the conversion of lanosterol to cholesterol in mammals or ergosterol in yeast. Thus the require- ment for oxygen cleanly divides the mevalonate pathway into an early and a late section, with squalene being the last product of the early oxygen-inde- pendent section (Fig. 1). The availabil- ity of oxygen is, thus, a critical determi- nant of the flow of molecules through the mevalonate pathway. Owing to the role of oxygen in the mevalonate path- way, the two independent controls on Hmglp production work in concert (Fig. 3): when oxygen availability is high, early mevalonate-pathway prod- ucts are low as a result of the efficient conversion into sterols by oxygen- dependent reactions, allowing post- translational Hmglp. High oxygen levels also directly stimulate the production of HMGI mRNA. When oxygen availability

is low, there is less efficient conversion of early mevalonate-pathway products to sterols and potentially greater pools of translation-inhibiting molecules. At the same time, the lower oxygen results in less stimulation of HMGI transcrip- tion. So the net effect, through two mechanisms, is high oxygen, high Hmglp; low oxygen, low Hmglp.

The synthesis of Hmg2p is also con- trolled by both the mevalonate pathway and oxygen. By contrast with Hmglp, the synthesis of Hmg2p is feedback regulated by molecule(s) that come from the late parts of the pathway and, thus, these signals require oxygen for their formation. Also, by contrast to HMGI, the transcription of the HMG2 gene is inhibited by high oxygen and stimulated in low oxygen. Again, the in- dependent features of feedback and oxygen control of Hmg2p synthesis can work in concert (Fig. 3): when oxygen is high, there is efficient production of late (post-squalene) products of the meval- onate pathway and consequently greater feedback inhibition of Hmg2p synthesis. At the same time, there is also oxygen- mediated repression of the transcrip- tion of the HMG2 gene and, thus, both regulatory effects work in the same di- rection. When oxygen availability is lowered, late mevalonate-pathway prod- ucts can not be made. The final result is a lessening of feedback inhibition of Hmg2p synthesis as well as direct stimu- lation of HMG2 gene transcription by lowered oxygen. The net effect of this concerted control is: high oxygen, low Hmg2p; low oxygen, high Hmg2p.

Owing to a less thorough understand- ing of the mechanism(s) of feedback control of Hmg2p synthesis, it is for- mally possible that the effect of oxygen on Hmg2p synthesis is mediated solely through effects on the flux through the mevalonate pathway. However, as other yeast proteins that are similarly regu- lated by the mevalonate pathway are not stimulated by lowering oxygen 32, we believe that independent controls exist.

A tale of two isozymes Why should yeast express Hmglp

during aerobiosis and Hmg2p during anaerobiosis? As described above, the two proteins display a striking differ- ence in post-translational regulation. The Hmglp protein is extremely stable and the Hmg2p displays rapid, regu- lated degradation. Hmg2p degradation is controlled by early signals from the mevalonate pathway 16 that do not re- quire oxygen for their synthesis. The

143

RiVIEWS TIBS 23. - APRIL 1996

High I

o x y g e n ~

buildup I

I I r

Hmg2p

H m g l p ~ I

synthesis high , I

Hmg2p synthesislow

Low oxygen

Hmglp synthesis low

Hmg2p synthesis high

Figure 4 Temporal view of the role of Hmg2p in acute and chronic oxygen loss. When an aerobic yeast cell (red) first encounters anaerobic conditions, the synthesis of Hmg2p increases. Although the synthesis of Hmglp slows (see Fig. 3), the extreme stability of Hmglp re- sults in high levels of each enzyme in cells that have undergone the transition from high to low oxygen (yellow). If oxygen is restored in a relatively short time, the synthesis of Hmg2p drops, and the yellow transition cell is restored to aerobic levels (red cell) of each isozyme by the rapid degradation of Hmg2p. If oxygen levels remain chronically low, the yellow transition cells will give rise to daughter cells (blue bud and cell) that have the anaerobic levels of each enzyme, as depicted.

rapid degradation of Hmg2p means that the steady state of this isoform is dy- namic. The size of the Hmg2p pool can be adjusted by altering either the syn- thesis rate or the degradation rate, as the steady state of a degraded protein is determined by the balance between synthesis and degradation. Furthermore, a rapidly degraded protein will attain a new steady state more rapidly when this balance is altered 36. Thus, in an- aerobic cells, HMG-R activity can be rapidly modulated in either direction by molecules from the mevalonate pathway that are made in anaerobic conditions.

These differences in the behavior of the HMG-R isozymes lead to a natural explanation of their differential synthe- sis. In a well-aerated yeast cell, all of the ergosterol that a cell needs is directly synthesized by the mevalonate path- way 37. Furthermore, the cell can safely store any excess ergosterol as inert acyl esters 3. The continuous synthesis and storage of ergosterol esters can be con- sidered a molecular sink for removal of excess early mevalonate products (such' as farnesyl groups). Experiments using drugs to inhibit the mevalonate pathway show that the Hmglp enzyme can only be modestly induced in these circumstances, indicating that the early pathway products that repress Hmglp synthesis are not abundant 27. Thus, in aerobic yeast, Hmglp is being synthe- sized 'full blast' and is totally stable; consistent with the constant need for sterols. There is little or no build up of early products that would reduce

Hmglp synthesis because of the 'sink' that sterol synthesis provides.

An anaerobic yeast cell presents a totally different picture. For clarity, con- sider the fully anaerobic cell. In this situation, the mevalonate pathway ends at squalene. Because the early pathway products and molecules derived from them are essential, the pathway must run, but now there is no molecular sink for any excess products. By contrast with inert, storable ergosterol esters, early pathway products such as squa- lene and farnesyl pyrophosphate have the potential to be cytotoxic because of the physical properties of the mol- ecules. Other early pathway products have been proposed to have direct ef- fects on cell growth 38,39. So a rationale for the favored synthesis of the Hmg2p isozyme emerges: in precisely the con- ditions where early pathway products can build up and cause harm, the cell synthesizes the HMG-R isozyme, which undergoes regulated degradation in re- sponse to these products. Regulated degradation, allows rapid adjustment of the balance between production of and cellular demand for the early pathway products in a situation where overabun- dance can not be tolerated.

Hmg2p as a stress protein In yeast cells exposed to transient or

acute reductions in oxygen, one would expect high levels of both Hmglp and Hmg2p. This is because the drop in oxygen causes increased synthesis of Hmg2p and, while Hmglp synthesis

drops, its level does not decrease be- cause of its high stability. Thus, the ceil, newly exposed to low oxygen (yellow in Fig. 4), has both isozymes in abundance. Continuous low oxygen levels result in the eventual produc- tion of daughter cells that have the an- aerobic complement of HMG-R isozymes: low Hmglp and high Hmg2p (blue cell). If, however, oxygen becomes available before new daughter cells are pro- duced, the synthesis of Hmg2p is halted and the existing Hmg2p is rapidly de- graded as a result of a high flux through the mevalonate pathway. The Hmg2p protein, thus, resembles a stress pro- tein that is specifically produced a t times of low oxygen tension and is de- stroyed when that stress is removed. If true, then this view implies that Hmg2p would he useful in times of transient oxygen deficiency. Perhaps its trans- membrane region has other functions that serve a cell under such stress. Because both mammalian and plant HMG-R synthesis are modulated during pathogen invasion 4,23,24,26, it is tempting to speculate that decreased oxygen level might serve as an indicator of en- vironmental invasion or overcrowding in yeast and that Hmg2p induction is a poorly understood response to this warning signal.

Study of HMG-R reveals that this es- sential enzyme is regulated in many ways. These modes of regulation in- clude multiple controls over the level and activity of a particular isozyme and separate control of multiple isozymes. It is clear that both the mevalonate pathway and independent biochemical processes can participate in the regu- latory cascade. Our studies in yeast demonstrate that the perplexing fea- tures of HMG-R structure and regulation can be understood in the context of the physiology of the whole organism. Furthermore, in studying the regulation of HMG-R, we find a way to understand both the regulation of the cholesterol pathway and a group of proteins that will be informative about integrated physiology, tissue-specific biochemistry and the conserved features of molecu- lar responses to stress and pathogens.

Acknowledgements This work was supported by grants

from the Helen Hay Whitney Foundation and the California Chapter of the American Heart Association (R. Hampton), by grants from the National Institutes of Health GM35827 (J. Rine), by an NIH training grant (T32GM07270)

144

TiBS 21 - APRIL 1996

and by a Mutagenesis Center Grant from the National Institute of Environ- mental Health Sciences (P30ESO1896-12). The authors wish to thank J. A. Watson for critical reading and helpful sugges- tions, and R. Zitomer for the obser- vation that Hmg2p could be considered a stress protein.

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Strategies for RNA folding

David E. Draper

RNAs are surprisingly adept at folding into specific shapes capable of ligand recognition and catalysis. Thermodynamic analysis of the unfolding of several different RNAs suggests that there are at least three strategies an RNA might use to achieve a very stable and compactly folded struc- ture: hydrogen bonding between irregular complementary surfaces (as in transfer RNA tertiary structure); monovalent and divalent ions bound to specific sites (as found in a ribosomal RNA fragment) and pseudoknot folds (exemplified by a messenger RNA fragment with extensive non- canonical structure).

A SURPRISE OF the past decade is the realization that RNAs readily adopt specific structures that are adapted, in their shapes and chemical properties, for molecular recognition and catalysis. A number of ribozymes have been de- scribed, and fairly short RNAs with high affinity and selectivity for small mol- ecules have been selected from random sequence pools. RNAs with nanomolar affinities for a variety of specific ligands (e.g. organic dyes, nucleotides and amino

D, E1 Draper is at the Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

�9 1996, Elsevier Science Ltd

acids) have been selected from pools of random sequence RNA. The ease with which such RNAs can be found implies that RNAs often fold into structures with unique and irregular shapes 1-3. RNA structural diversity is evidently compar- able to that of proteins, an unexpected result for a molecule with only four 'side chains'.

RNAs form regular and stable helical secondary structures from canonical base pairing; additional non-canonical and 'tertiary' interactions can give RNA an irregular shape (a definition of 'ter- tiary' is discussed below). Transfer RNA (tRNA), with its familiar set of inter-

PII: S0968-0004(96) 10021-9

actions between loops of the cloverleaf secondary structure, is currently the only RNA tertiary fold for which both atomic resolution structure and stabil- ity are known in detail. As the variety of tertiary and non-canonical interactions is presumably what accounts for the di- versity of RNA structure, it is pertinent to ask how extensive and stable such additional interactions might be in dif- ferent RNAs. This is not a difficult ques- tion to pose experimentally: the sec- ondary structure of an RNA can be reliably determined by phylogenetic comparisons and site-directed mutations; any differences between the actual folding energetics and the stability ex- pected for the secondary structure alone must arise from additional inter- actions of interest 4. Results from recent studies of this sort have suggested that stable tertiary folds could be achieved by several different means. To contrast these different strategies, this review summarizes the energetics of forming tRNA tertiary structure, of folding a ribosomal RNA structure dependent on specific ions and of folding a messenger RNA pseudoknot.

tRNA secondary and tertiary structure stability

Studies in the 1970s by several groups established that certain tRNAs unfold in a few discrete steps as the temperature is raised and that a set of

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