Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins

et Brbphyska &a

ELSEVIER

Biochimica et Biophysics Acta 1212 (1994) 259-277

Branch-point

reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins Jacob Griinler a, Johan Ericsson a, Gustav Dallner alb~* aDepartment of Biochemistry, University of Stockholm, Stockholm, Sweden b Clinical Research Center, Novum, Karolinska Insritutet, S-141 86 Huddinge, Sweden (Received 1 December 1993; accepted 16 March 1994)

Key words: Cholesterol; Dolichol; Ubiquinone;

Prenylated

protein

Contents 259

..................................................

1. Introduction

2. The mevalonate pathway ...........................................

260

3. The branch-point reactions ..........................................

261

4. Geranylgeranyl pyrophosphate synthase ...................................

263

5. Protein Isoprenylation .............................................

264

6. &Prenyltransferase

267

..............................................

270

7. Squalene synthase ............................................... 8. trans-Prenyltransferase

............................................

271

9. Subcellular distribution

............................................

272

10. Regulation

273

...................................................

Acknowledgements

............

; ....................................

274

References ......................................................

* Corresponding author. Fax: +46 8 7795585. Abbreviations: HMG, 3-hydroxy-3-methylglutaryl; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranylpyrophosphate; PFT, protein: FPP transferase; PGGT, protein: GGPP transferase; FPS, farnesyl-PP synthase; GGPS, GGPP synthase; LDL, low-density lipoprotein; ER, endoplasmic reticulum: SCP, sterol carrier protein; SRE, sterol regulatory element; DEHP; di(2-ethylhexyI)phthalate. 0005-2760/94/%07.00 0 1994 Elsevier Science B.V. All rights reserved SSD10005-2760(94)00051-Y

274

1. Introduction The initial portion of the mevalonate pathway supplies substrate for the highly-branched sequence of reactions leading to the formation of polyisoprenoid lipids such as cholesterol, dolichol and ubiquinone, as well as of isoprenoids covalently linked to protein, heme and tRNAs. In the initial portion of this path-

260

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way, three acetyl-CoA units are condensed to 3-hydroxy-3-methylglutaryyl CoA (HMG-COA), which is then further metabolized to isopentenyl pyrophosphate (IPP), i.e., the basic five-carbon isoprenoid unit. Sequential condensation of three IPP units gives rise to farnesyl pyrophosphate (FPP). According to the established concept, FPP is the common substrate for the so-called branch-point enzymes, i.e., the enzymes catalyzing the first committed steps in the biosynthesis of cholesterol, dolichol, ubiquinone and isoprenylated hemes and proteins. To date none of the enzymes specific for dolichol or ubiquinone biosynthesis and only a few of those participating in cholesterol biosynthesis have been purified from mammalian sources. Recently, however, new information concerning the biosynthesis of FPP and geranylgeranyl pyrophosphate (GGPP) and, especially, concerning the enzymes which utilize these two isoprenoids for protein isoprenylation and further lipid biosynthesis has appeared. It is now clear that the pattern of branch-point reactions is more complex than was originally believed and that a number of new enzymes must be included in this model. According to the original concept, the common cytoplasmic pool of FPP is the substrate for cholesterol and dolichol biosynthesis on the endoplasmic reticulum (ER), as well as for ubiquinone biosynthesis in mitochondria. This concept has now been modified by two observations: first, the biosynthesis of polyisoprenoid lipids appears to occur not only in the ER, but also in other organelles, such as peroxisomes, mitochondria and Golgi membranes; and secondly, the discovery that a large number of proteins contain covalently bound isoprenoids such as farnesol and geranylgeraniol, has substantially increased the number of enzymes and cellular processes known to be dependent on the production of FPP. In the present review we attempt to summarize new developments concerning the branch-point reactions of the mevalonate pathway and to discuss the complex regulation of these reactions, as well as the effects of such regulation on the production of the various endproducts at various cellular locations. Since both the initial and terminal steps in the biosynthesis of the different lipid end-products of the mevalonate pathway have been discussed in great detail in a number of recent reviews [l-9], these reactions will only be mentioned briefly here. 2. The mevalonate

pathway

The first reaction in the mevalonate pathway is catalyzed by the cytosolic enzyme acetoacetyl-CoA thiolase which condenses two acetyl-CoA units to give acetoacetyl-CoA (Fig. 1). In the next reaction, catalyzed by HMG-CoA synthase, acetoacetyl-CoA is condensed with an additional molecule of acetyl-CoA to

Acetyl-CoA 1 AcetoacetyCCoA 1 HMG-CoA HMG-CoA

tMevalonate 1 Mevalonate-P

shunt

Isopentenyl-tRNA

1 Mevalonate-PP 1 Isopentenyl-PP

reductaee

Mitochondrial

1 ---

1 Dimethylallyl-PP

1’

I 40Hbenzoate J Ubiquinone

Fig. 1. Organizationof

1 Cholesterol

the enzymes

/ Dolichol

of the mevalonate

\ 1 Dolichyl-PP

pathway.

give HMG-CoA [lo]. These two cytoplasmic reactions are followed by a reaction mediated by an integral ER protein, HMG-CoA reductase. In this process HMGCoA is reduced to mevalonate in a two-step reaction, utilizing two molecules of NADPH [11,12]. The structure of the intermediate formed in this reaction is of considerable interest, since it is very similar to those of pharmacologically important inhibitors of the reductase. Since HMG-CoA reductase is considered to catalyze the regulatory step in cholesterol biosynthesis and may also regulate the synthesis of other end-products of the pathway, this enzyme has been well characterized and we have extensive knowledge about its structure, biosynthesis and function [13,14]. The subsequent enzymes, responsible for the production of FPP, are localized primarily in the cytosol. This subcellular organization is somewhat unusual, but the complex regulation of HMG-CoA reductase appears to depend on its membrane association. Mevalonate kinase and phosphomevalonate kinase mediate the sequential phosphorylation of mevalonate to give mevalonate 5_pyrophosphate, utilizing two molecules of ATP [15]. Mevalonate pyrophosphate decarboxylase, which is also an ATP-requiring enzyme, catalyzes the dehydration-decarboxylation of mevalonate pyrophosphate [16,17]. The product of this reaction, isopentenyl pyrophosphate (IPP), is not only an intermediate in the pathway, but is also a required substrate for a large number of the subsequent elongation reactions involved in the biosynthesis of polyisoprenoids [lS]. IPP isomerase catalyzes the isomerization of IPP to form dimethylallyl pyrophosphate (DMAPP) [19]. FPP synthase mediates the sequential condensation of DMAPP with two molecules of IPP to give rise to FPP, the last common intermediate in the mevalonate

J. Griinler et al. /Biochimica

mtein erase

cidrenyltransferase

I

I

Fig. 2. Branch-point

enzymes

in the mevalonate

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et Biophysics Acta 1212 (1994) 259-277

pathway.

pathway [20]. The geranyl pyrophosphate (GPP) formed in the course of the overall reaction sequence appears to exist only as an enzyme-bound intermediate [21]. However, in a number of processes GPP may be the initial substrate [22]. In addition, DMAPP is also involved in the isopentenylation of certain tRNA species [23,24]. An additional sequence of reactions related to this pathway are the mitochondrial reactions which convert DMAPP back to HMG-CoA [25]. Further metabolism of this HMG-CoA results in the formation of acetylCoA and ketone bodies. This mitochondrial shunt may be involved in the removal of excess substrates from the mevalonate pathway.

the cytoplasmic fraction, but recent investigations have demonstrated that this enzyme activity is also present in other cellular compartments. In hepatic mitochondria the enzyme is associated with the mitochondrial matrix, where its specific activity is l/6 that of the cytosol [32]. These figures indicate that as much as 13% of the total cellular capacity to produce FPP may be associated with mitochondria in rat liver. The detailed fate of FPP synthesized in mitochondria remains to be established. However, it might be utilized for isoprenylation of heme and/or proteins or for the production of solanesyl and decaprenyl pyrophosphate. To a somewhat lesser extent FPP is also produced within peroxisomes and may thus be utilized by the squalene synthase and cis-prenyltransferase activities present in this same organelle [33]. Low FPP synthase activity is also associated with extensively washed microsomes and is not due to cytosolic contamination [34]. Fig. 3 shows Western blots of cytosolic, mitochondrial, microsomal and peroxisomal proteins using antibodies against rat liver FPP synthase. In vivo membrane-associated FPP synthase activity may have a considerable impact on the synthesis of lipid end-products, even in comparison with the cytoplasmic IPP pool. Thus, it is possible that the FPP generated within organelles is preferentially used in various biosynthetic reactions. FPP synthase consists of two identical subunits, each with a molecular weight of approx. 39 kDa [21,35-371. Both subunits contain one allylic and one homoallylicbinding site. As is the case for many of the condensing

3. The branch-point reactions The production of FPP plays a central role in the mevalonate pathway since FPP is the last common substrate for the synthesis of all the end-products (Fig. 2). FPP synthase has been purified from a number of sources and characterized in great detail [26-311. It catalyzes the sequential l’-4’ condensation of IPP with DMAPP and GPP in the truns-configuration in order to give all-trans FPP. FPP represents the last cytosolic intermediate of the pathway and the subsequent enzymes capable of utilizing FPP for the synthesis of cholesterol, dolichol and ubiquinone are membranebound. Although no investigations concerning possible interaction of FPP synthase with different membranes and proteins have yet been performed, direct interactions between this cytoplasmic enzyme and the membrane-enzymes utilizing FPP as substrate, i.e., squalene synthase and cis- and truns-prenyltransferase may well occur. The cytosolic protein : FPP transferase (PFT) may also be involved in direct protein-protein interaction with FPP synthase, in order to ensure a sufficient supply of substrate even when the overall flow through the pathway is down-regulated. The major site of FPP synthesis in eukaryotic cells is

FPP synthase (39 kDa)

CYT

MIT

PER

MIC

Fig. 3. Western blots using anti-FPP synthase antibodies (courtesy of Dr. P.A. Edwards, UCLA, Los Angeles). Lane 1, cytosolic; lane 2, mitochondrial; lane 3, peroxisomal; lane 4, microsomal protein.

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et Biophysics Acta 1212 (1994) 259-277

Table 1 Enzymes containing the putative allylic-binding site Enzyme

Species

Ref.

FPP synthase

Human Rat S. cerevisiae

41 42

GGPP synthase Phytoen synthetase Squalene synthase Hexaprenyl pyrophosphate synthetase Parahydroxybenzoate: polyprenyl transferase p subunit of protein : FPP transferase

E. coli N. crassa R. capsulatus E. herbicola S. cerevisiae S. cerevisiae

43 44 45 46 47 48 38

S. cerevisiae

39

Rat

4930

enzymes of the mevalonate pathway, FPP synthase requires Mg2+ or Mn2+ for activity and exhibits an ordered sequence of substrate-binding, with the allylic substrate-binding first. The amino acid sequences of FPP synthase from a number of sources are known and four conserved domains have been identified [38,39]. Two of these regions are characterized by an aspartate-rich repeat with a consensus sequence of XDDXXD. The third conserved domain is similar to the active-site domain reported previously by Brems et al. [40]. The fourth region, located at the carboxyl terminus, is rich in basic residues. One of the aspartate-rich domains (domain II> has also been identified in several other enzymes known to utilize allylic isoprenoid pyrophosphate substrates (Table 1). Based on these findings, together with the fact that site-specific mutagenesis of aspartate residues within this region strongly affects FPP synthase activity, it has been proposed that domain II represents the allylic-binding site of these proteins [51,52]. Using this sequence as a probe, it may be possible to identify the mRNAs for the as yet not characterized enzymes cis- and truns-prenyltransferases in the future. FPP synthase isolated from pig liver exists in two interconvertible forms, A and B 153-551. There is no difference in amino acid composition, but the A form possesses six free SH groups, while the B form contains only four such groups. Furthermore, the B form has a greater negative surface charge than does the A-form. It is possible that the difference in surface charge between these two forms of the enzyme may be important for the protein-membrane or protein-protein interactions that FPP synthase theoretically may be involved in. FPP synthase activity in liver is regulated by factors known to regulate the biosynthesis of cholesterol, such as fasting, cholesterol feeding and treatment with mevi-

nolin or cholestyramine. Most experiments have demonstrated coordinated regulation of FPP synthase, HMG-CoA synthase and HMG-CoA reductase activities. Recent experiments have demonstrated that following mevinolin treatment peroxisomal FPP synthase activity increases in parallel with the cytosolic activity, while the mitochondrial activity is unaffected (see Ref. 32 and Andersson, M. and Ericsson, J., unpublished results). These experiments indicate that the mitochondrial enzyme may be under separate regulation. Administration of peroxisome proliferators, such as clofibrate and, especially DEHP, induce peroxisomal FPP synthase activity, whereas the corresponding cytosolic and mitochondrial activities are not substantially affected. Since treatment of rats with these compounds increases the number of peroxisomes several fold, the overall effect on the peroxisomal activity is even greater, i.e., the increase in total peroxisomal FPP synthase activity is almost 50-fold compared to the control value. After cholestyramine treatment, there is a 13-fold increase in the cytosolic activity, but only limited change in the corresponding peroxisomal or mitochondrial activities. These results indicate that not only mitochondrial, but also peroxisomal FPP synthase activity may be regulated independently of the corresponding activity in the cytoplasm. In comparison with cytosolic and mitochondrial activities, peroxisomal FPP synthase activity is low. However, this activity is sufficient to supply allylic substrates for peroxisomal cis-prenyltransferase and squalene synthase. Under normal conditions, these two latter activities account for as much as 25 and 12% of the total hepatocellular activities, respectively. The compartmentalization of the FPP synthase might allow regulation of separate pools at different locations, but this possibility has not yet been investigated. In rat liver at least five sequences with high homology with that of FPP synthase are present in the genome, an indication that several transcripts might exist [56]. However, at least two of these sequences are processed pseudogenes 1571 and, furthermore, data suggest that the promotor region of the FPP synthase gene is present in a single copy [58]. The gene for FPP synthase contains two promotors [56]. One of these is activated in testicular tissue, while the other seems to be activated in all other cell types. Data are available which indicate that the promotor region of a rat genomic clone coding for FPP synthase contains 5 copies of the sterol regulatory element SRE1. This sequence is also found in the promotors for the genes for HMG-CoA synthase [591, HMG-CoA reductase [60] and the LDL receptor 1611 and regulate the expression of these genes in response to sterols. Although mRNA levels for FPP synthase are induced 4-fold in the absence of sterols, this induction does not

J. Gtinler et al. /Biochimica

involve SRE-1. The mechanism for regulation important gene awaits further elucidation. 4. Geranylgeranyl

pyrophosphate

263

et Biophysics Acta 1212 (1994) 259-277

of this

synthase

Until recently the synthesis of all-truns-GGPP in animal cells has received only limited interest, since the function of this product was not known. This situation is now completely changed, since isoprenylation of proteins with GGPP has turned out to be both a common and very important regulatory process. In the case of plants and photosynthetic bacteria, however, it is well established that all-trans-GGPP is utilized for the synthesis of carotenoids, the phytol moiety of chlorophyll and the side-chains of quinones and tocopherols, as well as in the biosynthesis of various diterpenes [62]. The partial purification of an all-truns-GGPP synthase from pig liver cytosol has been described [63]. Because of the low enzyme activity present in this preparation, no further characterization could be performed. Subsequent studies demonstrated that mevalonate could be utilized for the geranylgeranylation of proteins by cytosolic fractions from a number of different sources, indicating indirectly that a GGPP synthase is present in the cytosol [64]. Recent investigations have shown that the cytosolic fractions from various rat tissues can produce substantial amounts of GGPP, in addition to FPP, from labeled IPP [65]. The proportions of these two products differ greatly, depending on the tissue employed, and Fig. 4 shows the product distributions using liver and brain cytosols, as the enzyme source. In liver the dominant product is FPP, while in brain FPP and GGPP are produced in about equal amounts. This latter finding is in good agreement

with the fact that certain mevalonate pathway lipids are synthesized at a relatively low rate in adult brain [66]. Consequently, only a limited portion of the newly synthesized FPP is utilized for lipid synthesis and a considerable portion is available for GGPP production and protein isoprenylation. In experiments with isolated rabbit reticulocyte cytosol and labeled mevalonate, it was demonstrated that biosynthesis of FPP and GGPP may be affected by the requirement for protein isoprenylation [67]. When the concentration of different protein acceptors is increased, the synthesis of the specific isoprenoid utilized to prenylate the added protein is specifically increased. Furthermore, addition of unlabeled GGPP specifically inhibits GGPP, but not FPP synthesis. Clearly, the syntheses of FPP and GGPP are regulated independently and the rates of synthesis may be modulated directly by functional requirements. This conclusion is supported further when the two synthase activities in rat liver cytosol are assayed after mevinolin treatment [68]. FPS activity increases 4-fold in the treated animals, whereas GGPS activity is somewhat decreased. GGPP synthesis takes place at several intracellular locations, such as the cytosol, ER, mitochondria and peroxisomes. The properties of the enzymes involved in the cytosol and in a total-membrane fraction were recently examined [68]. Under the conditions employed, the membrane-associated enzymes produce truns,[email protected], while the cytosolic enzyme produces only the all-truns isomer (Fig. 5). The latter activity was found to be considerably higher in rat brain, spleen and testis than in liver (Table 2). The cytosolic GGPP synthase activities in different rat tissues are highly activated by Zn*+, have a narrow pH optimum around 5-6 and, in rat brain, the apparent

G

F

GG

G

F

I

I

I

I

I

GG

I

1

I ’ I 10

Retention time (min)

20 Retention time (min)

Fig. 4. Separation of the reaction products formed from 13H]IPP by cytosolic fractions. (A) Liver and (B) brain cytosol. The arrows indicate the elution of G: geraniol, F: farnesol and GG: geranylgeraniol standards. Taken from Ref. 53.

J. Griinler et al. / Biochimica et Biophysics Acta 1212 (1994) 259-277

264

all-tnl”,-GGPP

trans,trams,cir-GGPP

Fig. 5. Reactions

catalyzed

by the two GGPP

K, value for FPP is as low as 0.6 PM. Interestingly, whereas most of the enzymes of the mevalonate pathway are inactivated by Zn2+, GGPP synthase and three protein : prenyltransferases demonstrate an absolute requirement for this divalent cation. In addition, there are theoretically four more GGPP synthases present in the cell, i.e., the ttc-GGPP synthase in microsomes and peroxisomes and the alltruns-GGPP synthases in the ER and mitochondria. During the course of dolichol synthesis in microsomes and peroxisomes, in fact, ttc-GGPP accumulates either as the product of a specific GGPP synthase using FPP as substrate and/or as an intermediate of the sequential cti-prenyltransferase-catalyzed condensation reaction [34,69]. Both the microsomal truns-prenyltransferase which utilizes GPP as substrate [22] and the mitochondrial enzyme with a similar function, but employing FPP as substrate [32], synthesize solanesyl- and decaprenyl-PP, and all-truns-GGPP should appear as an intermediate. This product, may, however, be enzyme-bound and not occur in free form. A comparison of the specific activities of GGPS with those of PIT and PGGT-I in brain cytosol raises a number of questions concerning the regulation of the biosynthesis and metabolism of FPP and GGPP in this tissue. The specific activity of GGPS is almost 100 times higher than those of PFT and PGGT-I (Table 3). This high capacity for GGPP synthesis may serve to supply the cell with sufficient levels of substrate for GGPP-protein transferase (PGGT) I and II, even under conditions where the total flow of metabolites

Table 2 Tissue distribution Tissue

of prenyltransferases

a Data protein

in animal cells.

through the mevalonate pathway is reduced. Alternatively, GGPP may be required for other, as-yet-unidentified cellular processes. Since GGPS competes with protein : FPP transferase (PFT) for FPP, a strict regulation of these two activities must exist under in vivo conditions. Such regulation might be accomplished by specific protein-protein interactions between the appropriate cytosolic synthases and transferases. Experimental demonstration that such specific interactions do or do not occur will be an important task for future research in order to explain the complex regulatory processes involved at this branch-point of the mevalonate pathway.

5. Protein Isoprenylation FPP is utilized in a number of cellular processes, one of which is the isoprenylation of certain proteins. Although all-trans-GGPP is a theoretical intermediate in the reaction catalyzed by trans-prenyltransferase during the synthesis of the side-chain of ubiquinone, the only established function for all-trans-GGPP in animal cells is as a substrate for protein isoprenylation. Investigations in recent years have established that all eukaryotic cells contain a variable, but large number of isoprenylated proteins. In certain cell types as much as 2% of the proteins expressed are prenylated, i.e., about 60 different proteins per cell [70]. The large majority of these proteins are modified with geranylgeranyl (C,,) moieties (Table 4). Through the action of specific protein : prenyl transferases, a thioether linkage between the isoprenoid donor (in the pyrophosphate form) and a C-terminal

a

Activity FPP synthase

Liver Brain Spleen Testis Kidney

synthases

365 56 29 81 24

b

GGPP synthase 76 341 387 385 249

b

cb-Prenyltransferase

Table 3 Specific activities of various FPP and GGPP These values were calculated

’

168 32 98 179 9

taken from Ref. 68. b pmol of IPP incorporated/Fg per h. ’ pmol of IPP incorporated/mg protein per h.

of

brain

cytosolic

enzymes

from Refs. 68, 80 and 83.

Enzyme

Activity (pmol/mg

GGPP synthase PFT PGGT-I

400 4 6

prot per h)

which

utilize

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et Biophysics Acta 1212 (1994) 259-277

Table 4 Sequence dependence

for protein isoprenylation

Sequence

Isoprenoid

Protein

Transferase

CAAM/S

FPP

FPP : protein transferase

CAAL

GGPP

GGCC CCSN XCXC

GGPP GGPP GGPP

Ras, lamin A and B, y subunit of transducin, rhodopsin kinase y subunit of heterotrimeric G-proteins, ras-like low-molecular weight GTP-binding proteins, such as rat, ral and G25K Rab lB, rab 2 Rab 5 Rab 3A (or smgp25a)

cystein residue in the acceptor protein is established [9,71-731. This initial modification may be followed by others, such as proteolysis and carboxymethylation, but these modifications vary between different proteins. Prenylated proteins may be divided into two different classes on the basis of their C-terminal sequences. The first group contains the tetrapeptide CX,X,X,. Usually, Xi and X, are aliphatic amino acids, but prenylation is not dependent on the exact nature of these amino acids. On the other hand, the variability of X, is restricted and the nature of this amino acid actually determines the type of isoprenoid modification of the cystein residue, If X, is a polar amino acid, such as serine or methionine, the protein or peptide will be farnesylated. Examples of such proteins are Ras (X, = S) and nuclear lamins (X, = M). When X, is a nonpolar amino acid, e.g., leucine or phenylalanine, the protein or peptide will be geranylgeranylated. Examples of this kind of protein include the y subunit of heterotrimeric G-proteins (X, = L) and brain G25K (X, = P). Recently, novel prenylation sequences in which the position of the cystein residue is not as restricted as described above have been identified in rab proteins [74-771. In these proteins the C-terminal sequences may be XXCC, XCXC, CCXX or CCXXX. In all of these cases the cystein(s) are modified with a geranylgeranol moiety. In contrast to the CX,X,X, sequences, these latter sequences are not sufficient to allow prenylation, but further as-yet-unidentified information in the polypeptide, distal to the C-terminus, is also required. To date posttranslational isoprenylation of proteins has only been studied extensively in the cytosol and three different transferases have been isolated from the cytosol of mammalian brain and yeast and characterized. The protein : FPP transferase (PIT) that recognizes CX,X, X, sequences in which X 3 is a polar amino acid is a heterodimeric protein composed of an (Y (49 kDa1 and /3 (46 kDa) subunit [50,78-801. The protein : GGPP transferase (PGGT-I), which recognizes CX,X,X 3 in which X, is L or P is also composed of one (Y(49 kDa) and one p (43 kDa) subunit [81-841. The (Y subunits of these two enzymes are

265

GGPP : protein transferase I

GGPP : protein transferase II GGPP : protein transferase II GGPP : protein transferase II

believed to be identical. The p subunit of PF’T is involved in the binding of the acceptor polypeptide and contains a conserved domain corresponding to the putative-binding site for allylic isoprenoids, which is also found in a number of other prenyltransferases and synthases. An additional PGGT (PGGT-II) was recently identified and partially purified [85]. This enzyme is responsible for the geranylgeranylation of rab proteins and consists of two tightly associated components, A and B. The latter component contains two polypeptides with molecular weights of 38 and 60 kDa and the function of these two polypeptides appears to be analogous to that of the heterodimeric PFT and PGGT-I. The single polypeptide in component A has been proposed to be involved in the recognition of polypeptide sequences up-stream from the C-terminal portion of the acceptor protein. These transferases require Zn2+ and Mg2+ for optimal activity. Mg2+ seems to be involved in the proper binding of the isoprenoid pyrophosphate, while Zn2+, which is tightly associated with the protein, is most probably required for the interaction between the transferases and the acceptor polypeptide, in analogy with the situation in metalloproteases. Although only three protein : prenyltransferases have been described, it is very probable that this number will increase in the future, considering the large number of proteins with different structures and functions that have been found to be isoprenylated. Furthermore, additional amino acid sequences serving as acceptors for prenylation may be identified in the future and this in turn, will probably lead to the identification of new prenyltransferases. Association of polyisoprenoids with proteins through linkages other than thioether bonds has also been reported. About one-third of the dolichyl-P in rat liver remains bound to proteins after lipid extraction, gel chromatographic separation and proteolysis followed by chromatography [86l. This association is sensitive to alkaline hydrolysis, but not affected by iodomethane or treatment with Raney-nickel. The lipids released are identified by HPLC as dolichyl-P with various chain length. A similar type of covalent binding of polyprenyl phosphates to protein is also seen in spinach [87].

J. Griinler et al. /Biochimica

266

WJ-Cyl-

TH

et Biophysics Acta 1212 (1994) 259-277

1”

C~IJAAX

FPRprotein transferase

HzN“““(3

’ -C&AAX Proteolytic removal of C-terminal AAX

W’J -=,-

YH

I W-$-OH

0

I

C-Terminal carboxyl methylation

C&s- OCH,

Hp--+---

0

I

IPalmitoylation

HJV--&-‘-

Cjw-$-OCHS 0

Mahue ras-protein Fig. 6. Isoprenylation and further processing of ras proteins.

Further, polyprenols 11-13 and phytol were found in a protein-bound form not involving a tioether linkage in spinach. These observations are strong indications that within the near future the field of protein isoprenylation will grow even larger. Certain proteins, such as the rus proteins, are subject to additional C-terminal modifications following prenylation (Fig. 6). The tripeptide down-stream of the prenylated cystein is removed by specific membrane-associated proteases [88-901. This step is usually followed by methylation of the new C-terminal cystein by a membrane-associated S-adenosine methionine-dependent enzyme [91-931. In some cases up-stream cys-

tein residues may be palmitoylated. The modified rus protein is finally translocated to the plasma membrane, where it regulates cell growth. In the case of rus proteins, prenylation has been identified as the critical step in both translocation and transformation, and modifications in the proteolysis and/or methylation steps usually do not seem to prevent membrane association [94-961. It was demonstrated that the prenylation process must be post-translational, since cells treated with mevinolin accumulate non-prenylated precursor proteins which can be prenylated in a cycloheximide-insensitive manner when labeled mevalonate is subsequently added to the medium [97,98]. In order to inhibit the prenylation of pro-p21ras, CHO cells must be exposed to a level of mevinolin which is 500-fold greater than that required to completely inhibit cholesterol biosynthesis. In addition, 25hydroxycholesterol is unable to inhibit the prenylation process. These results suggest that the isoprenoids formed in the presence of these inhibitors are utilized preferentially for protein prenylation rather than for cholesterol synthesis. Protein isoprenylation seems to be involved in the targeting and membrane-binding of a number of proteins [991. G-proteins are required for intracellular signal transduction between receptors and effector enzymes. In many cases, one or two of the subunits of these heterotrimeric proteins are isoprenylated and this modification is often required for association of the functional complex with the membrane. Prenylation of nuclear lamins is also required for the proper assembly of these proteins into the nuclear lamina. Intracellular vesicular transport of proteins through the ER-Golgi system to the plasma membrane and other organelles and vice versa is an active process in many cells. Isoprenylated rab proteins are involved at several levels in this transport machinery. If prenylation of these proteins is prevented, vesicular transport may be stopped. Recently it has become clear that isoprenylation may also be of importance for direct protein-protein interactions. One example of this is the demonstration that the interaction between the a subunit and the b-g subunits of transducin is much more favored if the g subunit is farnesylated and carboxymethylated [loo, 1011. It was recently demonstrated that the (Y and p subunits of retinal cyclic GMP-phosphodiesterase are isoprenylated with a farnesyl and geranylgeranyl moiety, respectively, and that this modification probably facilitates association of these proteins with the outer segment disc membranes of the rod photoreceptor [102]. This latter suggestion has recently recieved additional experimental support [1031. These observations will surely prove important for future research which aims to identify specific receptors for prenylated proteins.

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Isoprenylation of proteins is an area of research which has only developed recently and, therefore, we may expect rapid progress in the near future. The major problems associated with investigations of protein isoprenylation are of a technical and analytical nature. Since isoprenylation occurs rapidly after protein synthesis, no detectable pool of non-isoprenylated proteins is present in cells. The life-time of the isoprenoid residue appears to be the same as that ot the polypeptide chain and, therefore, no exchange reactions seem to occur. The limited amount of lipid present on isoprenylated proteins does not allow direct chemical measurement and quantitation is based on incorporation of radioactivity. Consequently, practically all our conclusions are based on the incorporation of radioactive isoprenoid precursors into newly synthesized proteins. This procedure requires the use of rapidly growing cells with the capacity to effectively translocate such isoprenoid precursors across the plasma membrane. For these reasons studies of protein isoprenylation have so far been restricted to cultured cells. This, in turn, limits the types of cells which can be investigated and, since cells in culture are often dedifferentiated and transformed, it is difficult to estimate the extent and importance of protein isoprenylation under normal in vivo conditions. Furthermore, isoprenylation is not restricted to proteins, but has also been described to occur with certain species of tRNA and heme. Therefore, we may expect a large increase, not only in the number of known prenylated proteins, but also in the number of isoprenylated nucleosides (e.g., mRNA) and protein-associated heme residues in the near future. Fig. 7. Autoradiographic appearance of protein - GGPP transferase activity in cytosol from spinach leaves. Incubations were performed with isolated spinach leaf cytosol and [3H]GGPP in the presence (A) or absence (B) of peptide acceptor (KCCIL). Incubation were performed as described earlier [104]. The concentrations were for GGPP 25 PM, for the peptides 80 FM and the incubation mixture contained 30 pg cytosolic protein. The reaction mixture was analyzed on silica TLC by developing in ethyl acetate/ pyridine/water/acetic acid (lo:5 : 1: 3). The numbers indicate the migration of geranylgeraniol (11, prenylated peptide (21, GGP (3) and GGPP (4). The lower arrow gives the origin and the upper arrow shows the solvent front.

Protein isoprenylation has to date been studied almost exclusively in yeast and cultured mammalian.

Recently, however, we have found that this process is also active in plants (Parmryd, I, unpublished results). Furthermore, in the cytosolic fraction isolated from spinach leaves we identified high PFT and PGGT-I activities using synthetic acceptor peptides (Fig. 7). Recently, PIT has also been identified and cloned in garden pea, using conserved sequences from the mammalian and yeast proteins as probes [105].

6. &Prenyltransferase cis-Prenyltransferase mediates the sequential cis-addition of IPP units, commencing with the addition of IPP to all-truns-FPP [106-1101. A number of subsequent IPP additions results finally in the formation of a family of long-chain, a-unsaturated polyprenyl pyrophosphates. In animal cells in vivo the chain-lengths of these products vary between 16 and 23 isoprenoid units. A number of further reactions are required in order to complete the synthesis of dolichol, dolichyl-P and dolichyl esters, processes which are shown schematically in Fig. 8. It has been proposed that in the case of dolichol synthesis the last isoprene unit added is isopentanol instead of IPP and that the consequent lack of a leaving pyrophosphate group terminates the condensation sequence [ 1111. Alternatively, polyprenylPP may be directly dephosphorylated to yield polyprenol [112]. The final step in dolichol synthesis is a-saturation, mediated by an cY-saturase [ill], and it has been pro-

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et Biophysics Acta 1212 (1994) 259-277

FPP IPP -

-

Transferase Lower K,

-

IPP IPP IPP -

I

Transferase II Higher K, SCPZ-dependent

I Polyprenyl,-PP

Ieopentenol

a-Saturese

/\ Polyprenol,,

Polyprenyl-P

I

I

I Dollchol

CTP-kinase Z

I Dolichyl-P

Dolichyl-FA

Dolichyl-FA

Fig. 8. Reactions

involved in dolichol

biosynthesis.

posed that this reaction requires the participation of a cytosolic protein [112]. This enzyme is probably lowered in carcinogenic tissues [113,114]. Dephosphorylation of polyprenyl-PP to the monophosphate, followed by cu-saturation is required for dolichol-P synthesis [115]. Interconversion of the free alcohol and phosphorylated forms may occur through the action of the CTP-mediated dolichol kinase [116,117] and dolichol phosphatase [118,119], but it appears that under normal conditions these processes occur to a very limited extent [120]. Activation of these enzymatic steps does, however, seem to occur during development and carcinogenesis [121-1231. One could argue that two sorts of reactions are actually involved in the overall process catalyzed by cis-prenyltransferse. In the first step there is cis addition of IPP to all-truns- FPP, whereas all following additions are of the ‘&-to-& type. There are, in fact, several lines of evidence supporting the existence of two different reactions, catalyzed by separate enzymes, or, at least of a two-step reaction. After a short in vitro incubation of microsomes with FPP and IPP, two products appear upon HPLC of the reaction mixture, i.e., GGPP and long-chain polyprenyl pyrophosphates [34,124,125]. The GGPP has been identified as the trans,trans,cis-isomer (Fig. 9). When this short incubation is followed by a chase-period in the presence of excess unlabeled IPP, the radioactivity in GGPP is transferred to long-chain polyprenyl py-

rophosphates [34]. This experiment demonstrates that GGPP is synthesized in detectable amounts under in vitro conditions and that is actually an intermediate in the reaction catalyzed by cis-prenyltransferase. This is the only intermediate observed during in vitro formation of long-chain products. When these experiments repeated using peroxisomes instead of microsomes, GGPP was also found as an intermediate [331. Furthermore, when microsomes are solubilized under certain conditions, it is possible to detect a specific tram, truns,cis-GGPP synthase activity in the solubilized fraction. Whether GGPP is formed under in vivo conditions remains to be investigated. The properties of cis-prenyltransferase have been studied mostly using microsomal membranes [69,1261291. This enzyme activity is dependent on Mg2+ and is inactivated by inorganic phosphates, especially pyrophosphates. Both pyro- and monophosphates are formed even in the presence of phosphatase inhibitors. Maximal enzyme activity requires high concentrations of detergent and l-2% Triton X-100 is usually employed in the assay system. The apparent K, values for IPP and FPP were found to be 4.4 and 25 PM, respectively, for the rat liver enzyme. It has been established that sterol carrier protein 2 (SCP2), which is present in the cytoplasm and in peroxisomes, is required for the conversion of lanosterol to

4

mb

2

; a4 ,x ._ .I> ti .I8 B a!

2

4 2

!

3

c 10

30

Time fkin)

Fig. 9. Products formed during pulse-chase labeling of rat liver microsomes with [3H]IPP. Incubations were performed in the presence of [3H]IPP and FPP. (A) A 4-min pulse with [3H]IPP; B, a 4-min pulse followed by a 20-min chase; C, a 4-min pulse followed by a 120-min chase. The numbers above the peaks indicate the number of isoprene units. Taken from Ref.34.

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Table 5 Properties of sterol carrier protein 2 Distribution: Function:

Deficiency

:

Consequence of deficiency :

Primarily in the cytoplasm and peroxisomes Cholesterol synthesis Cholesterol esterification Dolichol synthesis Steroid hormone synthesis Lipid transport Peroxisomal diseases Malign tumors Nieman-Pick disease, type C Accumulation of cholesterol in specific intracellular organelles

cholesterol, for the activation of acyl-CoA: cholesterol acyltransferase and for pregnenolone biosynthesis [130] (Table 5). Most interrestingly, it was found that SCP2 is able to replace detergents in activating cis-prenyltransferase activity [1311. Maximal activation is obtained using as little as 1 pg purified SCP2. In contrast to detergents, the presence of SCP2 stimulates the formation of polyisoprenoid pyrophosphates with chain-lengths very similar to those found in vivo. Therefore, it appears probable that the endogenous activator of cis-prenyltransferase is SCP2. SCP2 may exert its effect by transporting substrates to the active site(s) of the enzyme and/or, alternatively, by the removal of the end-products. The activation of cis-prenyltransferase by SCP2 seems to be specific, since this protein has no effect on a number of other enzymes involved in isoprenoid metabolism. In spite of the fact that there have been a number of attempts to purify cis-prenyltransferase, successful purification has not yet been reported. The main problems are probably the lack of a simple and, even more important, rapid enzyme assay, as well as the apparent instability of the enzyme. Further developments in this field require successful purification of cis-prenyltransferase. Dolichol biosynthesis is not restricted to microsomes but is also recovered in peroxisomes [132]. Accordingly, cis-prenyltransferase activity is present in this organelle as well [33]. In fact, this specific activity in peroxisomes is 3-fold higher than that in microsomes. The substrate specificities in microsomes and peroxisomes are very similar, but the two activities demonstrate different requirements for detergents. In contrast to the microsomal activity, the peroxisomal enzyme is inhibited by detergents. One possible explanation for this observation may be that peroxisomes contain sufficient amount of SCP2 for maximal activation of cis-prenyltransferase without addition of detergents [133,134]. One way to differentiate between the peroxisomal and microsomal cis-prenyltransferase activities may be to measure the effects of treatments with different

269

agents on these activities. The peroxisome proliferators clofibrate and DEHP elevate the microsomal, but not the peroxisomal activity (Andersson, M. and Ericsson, J., unpublished results). On the other hand, mevinolin treatment increases the peroxisomal, but not the microsomal activity. Phenobarbital, a classical proliferator of the ER, increases the activity at both locations, but more extensively in microsomes. These studies indicate that the transferase may be independently regulated at these two different locations. Approximate calculations which take into consideration the numbers of different organelles in hepatocytes indicate that as much as 25% of the total rat hepatic cis-prenyltransferase activity may be associated with peroxisomes. After specific induction (e.g., by DEHP or clofibrate treatment) this organelle may become the major site of cis-prenyltransferase activity in hepatocytes. This is a very interesting conclusion, since it would indicate that peroxisomes may be the major site of dolichol biosynthesis under certain pathological conditions. It also raises a number of questions concerning the transport and function(s) of different dolichol derivatives. It will be an important future task to examine the subcellular localization of cis-prenyltransferase and dolichol biosynthesis in more detail and to determine how changes in transferase activity at different locations contribute to modifications in dolichol biosynthesis and function(s). The products of cis-prenyltransferase consist of a family of polyisoprenoid pyrophosphates containing different numbers of isoprene units. This pattern is species specific, but does not vary substantially between different cells within the same animal [135]. At present, the functional significance of a specific polyisoprenoid distribution and the broad product specificity of ci.s-prenyltransferase remain unexplained. If detergents are used in in vitro incubations, the chainlengths of the products are shorter than those found in dolichol derivatives in vivo (Table 6). Increased concentrations of mevalonate and/or IPP result in the

Table 6 In vitro and in vivo factors which regulate the chain length distribution of polyisoprenoids In vitro:

In vivo:

Substrate concentration (a) mevalonate (b) IPP (cl FPP Detergents SCP2 Treatment with inhibitors of HMG-CoA reductase DEHP treatment Hepatic hyperplastic noduli Tumor cells in tissue culture Human hepatocellular cancer Nieman-Pick disease, type C

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formation of longer polyisoprenoids, while increased concentrations of FPP results in the formation of shorter polyisoprenoids [69,111,124,125]. It is yet not clear why substrate concentrations well above those required to saturate the enzyme affect the lengths of the polyisoprenoids. As mentioned earlier, the addition of SCP2 to in vitro incubations leads to the formation of polyisoprenoids similar in length to those found in vivo. The importance of SCP2 for the formation of a normal isoprenoid pattern is further demonstrated by the peroxisomal system, in which the endogenous presence of this protein enchances the formation of polyisoprenoids with the correct chainlengths. There are certain conditions under which modifications of the polyisoprenoid distribution pattern have been reported. Treatment of rats with DEHP leads to the accumulation of longer dolichols [136], while the administration of mevinolin leads to the formation of shorter polyisoprenoids [137]. In hyperplastic noduli in rat liver the relative distribution of longer polyisoprenoids is elevated [138] and in human hepatocellular carcinoma [113] and mononuclear leukocytes from leukemic patients [139] the shorter chain-lengths dominate. Longer dolichols are also preferentially synthesized in Niemann-Pick disease, type c (Schedin, S., unpublished results).

7. Squalene synthase Squalene synthase catalyzes the first committed step in the biosynthesis of cholesterol, i.e., the ‘head-tohead’ (l’-2-3) condensation of two molecules of FPP (Fig. 10). This reaction occurs into two steps: first, the formation of presqualene diphosphate, which is then reductively rearranged in a second step to form squalene, using NADH or NADPH as the reductant. This enzyme was initially isolated from yeast [140] and only recently has a proteolytic fragment of squalene synthase which retained enzyme activity been isolated from rat liver microsomes [141]. The molecular weight of the non-processed protein is 45-47 kDa and it contains a single polypeptide chain. The enzyme has now been cloned from both Saccharomyces cerevisea [142] and rat liver [143], which allows a more detailed analysis of the protein. From the amino acid sequence of the rat liver protein it has been concluded that the

PPP

Pmqlulcnc pymphaphate

Fig. 10. The reaction

catalyzed

by squalene

sqrulen synthase.

protein probably contains three membrane-spaning regions, a conclusion which, however, disagrees with the data obtained from trypsin fragmentation. Three highly conserved domains in the rat liver and yeast enzymes have been identified. One of these sequences demonstrates a high degree of homology with the putative allylic-binding site found in a number of functionally distinct proteins which utilize polyprenyl pyrophophate substrates. It will be interesting to see whether these conserved sequences may be used in the future to ‘fish out’, for example, cis-prenyltransferase. Although HMG-CoA reductase is considered to be the major regulatory enzyme of cholesterol biosynthesis, squalene synthase may play a regulatory function under certain conditions, since it catalyzes the first committed step in cholesterol biosynthesis and therefore determines the flow of FPP into the membrane-associated portion of this pathway As is the case for HMG-CoA reductase, squalene synthase is down-regulated by LDL-cholesterol [144] and up-regulated by treatments known to up-regulate the biosynthesis of cholesterol, e.g., mevinolin and cholestyramine [141, 1451. In hepatocytes cholesterol biosynthesis has been recovered not only in the ER, but also in peroxisomes and, accordingly several enzyme activities of this biosynthetic pathway have been detected in this latter organelle [146-1481. Squalene synthase in peroxisomes is similar to its microsomal counterpart which, however, does not exclude the presence of two different proteins [33]. It requires Mg2+ for activity and the reduction of presqualene diphosphate to squalene is dependent on either NADH or NADPH. The specific activity of squalene synthase in peroxisomes is equally high as that in microsomes and in rat liver as much as 12% of the total hepatocellular activity may normally be ascribed to peroxisomes. Under conditions where the number of peroxisomes in the cell is induced, this figure may even be much higher. The peroxisomal squalene synthase is of special interest since in opposite to the microsomal enzyme, it is not downregulated in cholesterol treated rats. Inhibition of HMG-CoA reductase with substances such as compactin, mevinolin or related compounds is an important clinical treatment of hypercholesterolemia. Since these drugs affect the biosynthesis of cholesterol at a point of the mevalonate pathway prior to the central branch-point, there is a risk that the biosynthesis of end-products other than cholesterol may also be affected. In fact, the ubiquinone levels in liver, muscle and heart decrease when rats are treated with mevinolin for a 3-week period [137,149]. Therefore, it is clear that agents which specifically inhibit cholesterol biosynthesis distal to HMG-CoA reductase and the branch-point may offer significiant progress in this area. A number of such inhibitors are

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et Biophysics Acta 1212 (1994) 259-277

available and have been used in experimental systems, but not as clinical drugs. Recently, inhibitors of squalene synthase, i.e., squalestatin-1 [150], Zaragozic acids [151] and isoprenyl l,l-biphosphonates [1521 have been isolated. The inhibition of squalene synthase activity by these compounds results in an accumulation of FPP. The effects of squalestatin-1 on fractions isolated from rat liver homogenate and on tissue culture cells were investigated (Thelin, A., Peterson, E., Hutson, J.L., McCarthy, A.D., Ericsson, J. and Dallner, G., unpublished results). Squalene synthase is inhibited by a 1 mM concentration of this substance, leading to an accumulation of FPP, but not of GGPP. No effect on the FPP- or GGPP-synthases, cis-prenyltransferase or protein isoprenylation was observed. There was a 4-fold increase in the incorporation of [3H]mevalonate into ubiquinone, but it remains to be established whether this increase reflects an elevated biosynthesis of the lipid, These substances may prove to be effective in lowering cholesterol synthesis in humans, although clinical use presupposes that the accumulation of FPP has no negative consequences on other branch-point reactions and that the drugs do not directly affect any other enzymes of the pathway. In any case, these inhibitors offer unique tools for controlling the overall flow of FPP and, thereby, provide good possibilities for studying the regulation of the branch-point reactions.

8. hwzs-Prenyltransferase In eukaryotic cells long-chain all-truns polyisoprenoid pyrophosphates are produced by the enzyme trans-prenyltransferase. In animal cells the main or exclusive function of these all-trans polyisoprenoids, containing nine or ten isoprene units, is to construct the side-chain of ubiquinone. However, in plant cells there are a number of compounds in addition to ubiquinone which contain all-trans polyisoprenoid chains (e.g., plastoquinones). The all-truns-polyisoprenoid-PP is condensed with 4-hydroxybenzoate and a number of subsequent modifications of the ring are required for the completion of ubiquinone (Fig. 11). Ring completion after condensation starts with C-hydroxylation, followed by O-methylation and decarboxylation. Two additional C-hydroxylations and one 0-methylation are necessary for the final synthesis of ubiquinone. This sequence has been elucidated in bacterial systems [4,153] and it is suggested that in animal systems the pathway is somewhat different, the first step after condensation being decarboxylation [154]. Originally it was thought that ubiquinone is present exclusively in the inner mitochondrial membrane, where its only function is to participate as a redox-component of the mitochondrial respiratory chain. During recent

Fig. 11. Terminal

reactions

in the biosynthesis

of ubiquinone.

years this concept has been modified somewhat since ubiquinone has been found to be present in all cellular membranes investigated [155,1561. Furthermore, it is now well established that ubiquinone has an additional function as an endogenously produced antioxidant. Ubiquinol, the reduced form of ubiquinone, is the active antioxidant [157], but the enzymatic machinery responsible for the reduction of ubiquinone outside mitochondria has not yet been identified. The major part of ubiquinone in both rat and human tissues is in reduced form in vivo [158]. Animal cells contain about lo-times more ubiquinone than cu-tocopherol (vitamin E) and the cell preferentially utilizes the former as an antioxidant [159]. In spite of the fact that all eukaryotic cells probably contain trans-prenyltransferase activity, only a few investigations to date have been concerned with characterization of this enzyme activity in animal cells. In most investigations it has been found that tissues and also isolated organelles contain an excess of the precursor ring 4-hydroxybenzoate. The polyprenyl-PP synthesized is thus often not detected in its free form, but rather condensed with the precursor ring. The truns-prenyltransferase activity of rat liver microsomes was investigated recently (22). This enzyme, like its counterpart in spinach microsomes [160] and yeast mitochondria [161], utilizes GPP, but not FPP as the allylic substrat. This is in contrast to the corresponding enzyme in Bacillus subtilis, Micrococcus luteus and liver mitochondria [32,162,163], which is specific for FPP. The microsomal enzyme has a pH optimum at 8.0 and is activated by Mn2+, Mgzf and certain detergents. The trans-prenyltransferase, in contrast to its cis counterpart, is not dependent on cytosolit protein factors. The former enzyme is present on the surface of the rough and smooth ER and delivers its product to the ER-Golgi system, where it is utilized

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for ubiquinone biosynthesis [164]. The participation of the ER-Golgi system in synthesis of the polyisoprenoid side-chain, as well as in further synthesis of ubiquinone and plastoquinone has also been demonstrated in plant systems [ 1601. Rat liver mitochondria contain both IPP isomerase and FPP synthase [32]. The FPP synthesized within mitochondria is probably required for various reactions utilizing this compound as substrate. The FPP synthase is present in the mitochondrial matrix, a trans-prenyltransferase utilizing FPP as substrate, was, associated entirely with the inner mitochondrial membrane. The products of this reaction are both nona- and decaprenyl-PP. In contrast to dolichol the lenght of the side-chain of ubiquinone varies to a much more limited extent. In eukaryotic cells there are ubiquinones with 6-10 isoprene residues, but in every species a single chain lenght dominates. In humans the side-chain in all tissues is predominantly decaprenol and only a small proportion (2-7%) of solanesol is present. In rat most of the ubiquinone contains 9 isoprene residues, but in some tissues, such as brain, spleen and intestine, onethird of the ubiquinone has a decaprenyl side-chain. It is not known what determines these chain-lengths, but in in vitro systems the lengths can be varied only within a narrow range (i.e., one isoprene unit). Such variation is achieved by altering the concentrations of Mg2+ and the substrates mevalonate, IPP and FPP [32,165,166]. The gene for all-trans-hexaprenyl diphosphate synthase has been identified and cloned from S. cerevisae [381. A respiratory mutant defective in the biosynthesis of ubiquinone [167] regained this abillity when transfected with a plasmid containing this gene. From the deduced amino acid sequence it was possible to identify the allylic-binding site also found in a number of other enzymes utilizing allylic isoprenoid pyrophosphate substrates.

9. Subcellular distribution The concept of multi-site localization of biosynthetic processes has developed relatively recently. For a long time strict cellular compartmentalization of enzymatic activities was considered to be the basis for cellular function. On the basis of cell fractionation studies, it has been established that the initial portion of the mevalonate pathway is localized in the cytosol. A common cytosolic pool of FPP then serves as substrate for cholesterol and dolichol biosynthesis in the ER and for ubiquinone biosynthesis in mitochondria. The development of new fractionation and analytical techniques, together with the use of selective membrane proliferators have led to the conclusion that the biosynthesis of polyisoprenoid lipids may involve the interaction of

several compartments and that the same molecule may be produced in more than one organelle. The final product of the cytosolic portion of the mevalonate pathway, i.e., FPP, is not only utilized by various membrane-associated enzymes, but together with all-trunsGGPP, serves as substrate for cytosolic protein :prenyltransferases. In addition, IPP is utilized for tRNA isoprenylation, but it is not yet clear whether this reaction(s) is restricted to the cytoplasm. The microsomal fraction has been used in almost all studies of cholesterol and dolichol biosynthesis and, therefore, our knowledge concerning these processes is derived almost exclusively from studies with these membranes. This membrane also has the capacity to esterify cholesterol. Obviously, the microsomal processes are dependent on cytosolic proteins such as SCP2. All other enzymes participating in the latter portion of the mevalonate pathway seem to be integral membrane proteins. The ER also possesses truns-prenyltransferase and the product formed is transferred to a separate microsomal compartment (smooth II), where the terminal steps in ubiquinone synthesis take place. This subfraction, which is also capable of esterifying dolichol, is probably related to the Golgi system, but the exact nature of this relationship remains to be established. Esterification of dolichol involves CoAactivated fatty acids [168]. Hydrolysis of dolichyl esters occurs in lysosomes and, also, the plasma membrane [169]. At the surface of hepatocytes facing the circulation, dolichol may be esterified by a transacylase which preferentially utilizes C, fatty acyl moieties of phosphatidylethanolamine [170]. This latter enzyme has been shown to be identical to the hepatic lipase [171]. Isolated mitochondria contain a high level of FPP synthase activity and some of the FPP produced is obviously utilized for the synthesis of solanesyl and decaprenyl pyrophosphates, which in turn may be utilized for mitochondrial ubiquinone synthesis. The significance of mitochondrial ubiquinone synthesis cannot be evaluated at present, since using present assay procedures the relative ratio of FPP:nona-to decaprenylPP synthesis is as much as 500: 1. FPP is also the substrate for heme isoprenylation (cytochromes a and a,) [172], a process that has received relatively little attention so far. Therefore, it will be an interesting future task to try to identify and characterize the mitochondrial enzyme(s) responsible for the prenylation of porphyrins. Since mitochondrial, as well as cytoplasmic tRNA species are isoprenylated [173,174], it is reasonable to assume that mitochondrial enzymes specific for this process exist. Since mitochondrial FPP synthase comprises about 13% of the total cellular activity, it will be important to study the metabolism of FPP in this organelle in more detail. Peroxisomes can be said to contain more complete synthetic machinery for the production of cholesterol

J. Griinler et al. /Biochimica

and dolichol, since this organelle also contains high levels of SCP2. A number of enzymes involved in the initial portion of the mevalonate pathway are also present in peroxisomes (e.g., acetoacetyl-CoA thiolase, HMG-CoA reductase, mevalonate kinase, IPP isomerase and FPP synthase) and it is probable that the complete pathway is present in this organelle. It is well known that peroxisomal membranes contain only a few integral proteins and that the majority of peroxisomal proteins are present in the soluble matrix. Many of the enzymes involved in isoprenoid biosynthesis seem, however, to be membrane-associated and it will be of interest to characterize these enzyme systems further. The large number of enzymes involved in cholesterol biosynthesis and the highly hydrophobic nature of the substances produced and utilized by these enzymes makes it unlikely that substrates and active proteins are distributed freely in the soluble peroxisomal matrix. Most probably, multiple-enzyme complexes are present in the luminal compartment, but tightly associated with the membrane. A combination of classical yeast genetics and the fact that it is possible to obtain peroxisomal proliferation in yeast may prove very informative in this respect. Furthermore, the use of mammalian cells defective in peroxisomal proliferation or certain peroxisomal functions may also prove very useful. The subcellular distributions of enzymes and substrates/products involved in the mevalonate pathway are summarized in Table 7. Clearly, this organization is very complex and one may well ask why the cell has evolved such a multi-site organization of isoprenoid biosynthesis and metabolism. It is very probable that the products formed within different cellular compartments have different functions. Cholesterol and dolichol produced in the ER are important constituents of membranes and lipoproteins. Their peroxisomal counterparts may, however, be associated with,

Table 7 Subcellular

distribution

of the mevalonate

Peroxisomes Acetoacetyl-CoA thiolase HMG-CoA reductase Mevalonate kinase IPP - cholesterol IPP - dolichol SCP2 Mitochondria IPP - FPP Farnesyl synthase trans-Prenyltransferase IPP - nona- and decaprenyl-PP Heme isoprenylation Isoprenylation of t-RNA

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for example, the production and transport of bile. If ubiquinone produced also within mitochondria it is most certainly utilized primarely in the respiratory chain in the inner membrane, while that produced within the ER-Golgi system may be redistributed in the cell, as well as to extracellular compartments, and act as an antioxidant. The mitochondrial biosynthesis, however, may be absent or very low, which indicates that the mitochondrial ubiquinone originates from the ER-Golgi biosynthetic system [l%].Some of the secondary sites of lipid biosynthesis may constitute a protective ‘back-up’ mechanism for the cell. Down-regulation, inhibition or selective damage of the enzymatic machinery at one location may be compensated for by an increased rate of the corresponding metabolism in another organelle, which could allow the cell to maintain its normal functions.

10. Regulation

Investigations performed during at least two decades have firmly established that the major regulatory enzyme of the initial pathway, as well as of the overall biosynthesis of cholesterol is HMG-CoA reductase. A number of dietary treatments and physiological conditions influence the reductase activity at several levels. The involvement of HMG-CoA reductase in the regulation of the biosynthesis of other mevalonate-derived compounds is, however, questionable. Exposure of a number of different experimental systems, most notably rapidly differentiating cells or tissues, to inhibitors of HMG-CoA or cholesterol biosynthesis has been demonstrated that the biosyntheses of ubiquinone and dolichol, as well as of isoprenylated proteins are regulated by processes distal to HMG-CoA reductase. Good candidates for such a regulatory role are the first committed steps in the biosynthesis of these com-

pathway

Cytosol Acetyl-CoA - FPP (but not HMG-CoA reductase) GGPP-synthase Protein-prenyltransferases SCP2 Isoprenylation of t-RNA

Endoplasmic reticulum HMG-CoA reductase FPP - cholesterol Cholesterol esterification FPP - dolichol trans-Prenyltransferase Farnesyl synthase Golgi Dolichol esterification Ubiquinone synthesis (terminal

part)

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274

PROTEIN

ISOPRENYLATION

very hsgh ‘3ffimty

1. high affmty GGP -

,

DOLICHOL

FPP

low affmy

htgh affimty \

;OLANESYL-PP

Fig. 12. Affinities of branch-point way for their substrates.

CHOLESTEROL

enzymes

in the mevalonate

path-

lary electrophoresis may prove to be very useful in this regard. As also mentioned above, the multi-site distributions of certain of the branch-point enzymes may have a regulatory function. In order to further investigate this possibility, the different enzymes, e.g, FPP synthase, squalene synthase and cis- and trans-prenyltransferases, must be isolated from their different subcellular locations. This would allow detailed investigations on whether the same enzyme may be regulated independently at different intracellular locations.

Acknowledgement

pounds, i.e., the enzymes utilizing FPP as substrate. This type of regulation may be achieved by several different mechanisms. One of the proposals concerning such branch-point regulation is known as the ‘flow diversion hyphothesis’ [23]. In this model regulation is supposed to be mediated by the different affinities of the different branchpoint enzymes for FPP (Fig. 12). This model predicts that squalene synthase should have a low affinity for FPP compared to &-and trans-prenyltransferases and the protein : prenyltransferases. This, in turn, would mean that increases and decreases in the pool of isoprenoid substrates (i.e., FPP) would only affect the synthesis of cholesterol, since the other branch-point enzymes are saturated even at low substrate concentrations. Since a number of the branch-point enzymes have not yet been purified from mammalian sources (e.g., tram- and cis-prenyltransferases), information concerning their substrate affinities is only approximate. Furthermore, almost no investigations have been performed that were designed to actually measure whether the available pool of FPP in the cytosol varies under different conditions known to affect the biosynthesis of isoprenoid compounds. The ‘flow diversion hypothesis’ predicts that there exists a common pool of FPP in the cytosol and that this pool is available for all the branch-point enzymes. Another possibility is, of course, that FPP synthase directly interacts with the different branch-point enzymes which utilize FPP at their different intracellular locations and thereby directs the flow of substrates through the different branches of the mevalonate pathway. In this case there would be no common cytosolic pool of FPP available to the different branch-point enzymes. Since a number of the FPP-utilizing enzymes at the branch-point have recently been purified, antibodies are now available and it will be possible to search for such protein-protein interactions in the future using cross-linking reagents. As mentioned above, it will also be important to directly measure the intracellular concentrations of different isoprenoid substrates. Recent developments in the field of capil-

The research in the authors’ laboratories was supported by grants from the Swedish Medical Research Council and the Swedish Cancer Society.

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