JOHN ARMSTRONG PROTEIN PRENYLATION

lrw0 fingers for membrane traffic To function in vesicle transport, Rab/Ypt proteins require

modificatioin with geranylgeranyl groups. One subunit of the human transferase responsible is encoded by the choroideraemia gene.

A family of small GTP-binding proteins, usually called the Rab or Ypt proteins, are involved in regulating dif- ferent stages of membrane traffic (see preceding article in this issue [ 1] >. These proteins are made in the cytosol, but must be able to interact with membranes in order to function. This property is conferred on the proteins by the addition of the 20.carbon isoprenoid derivative, geranylgeranyl, to cysteine residues at or near their car- boxy1 termini. Seabra et al. have now purified the trans- ferase that causes this reaction [21. Unlike other protein isoprenyl transferases characterized so far, this enzyme has three subunits, not two. The third subunit, actually known as component A, h:as been partially sequenced. Remarkably, component A appears to be the product of a previously identified gene, defects in which cause the inherited disease choroideraemia - a form of reti- nal degeneration. This discovery is entirely unexpected, and not immediately explicable.

The first report of protein modification by prenylation came from work on fungal mating factors [3]. A 15.carbon farnesyl group, representing three isoprenoid ‘blocks’, was found attached by a thioether linkage to the sul- phydryl group of a cysteine side chain. This proved the key to understanding the modiEcations that occur on a somewhat more fashionable molecule, the Ras oncogene product [4]. This protein is translated with a so-called CAAX box (where A is an aliphatic amino acid residue and X is any amino acid) at its carboxyl terminus. After the cysteine side chain receives the farnesyl group, the three terminal residues are removed and a methyl group is attached to the newly-exposed carboxyl terminus. These alterations contribute to the protein’s ability to attach to membranes. The CAAX box, or near variants, has since been found on a variety of otherwise unrelated proteins, with the same consequences as for Ras.

The Ras protein, as well as clontinuing to attract much in- terest in its own right, has in the past few years become a phylogenetic grandfather. 1;t heads a superfamily of pro- teins in the 20-30kD size range that bind GTP, but can only hydrolyse it in concert with a GTPase-activating pro- tein (GAP) [5]. Family memlbers permeate many aspects of a cell’s activity. Some have a CAAX box in which the X is hydrophobic; in these cases the prenyl group that becomes attached is the 20carbon geranylgeranyl. The enzymes that attach the prenyl groups - the farnesyl and geranylgeranyl transferases -- are both dimeric, sharing a common subunit (reviewed ia [6]).

Members of another branch of the superfamily have a dif- ferent cysteine-containing structure, generally either CC or

CXC, at their carboxyl termini. These proteins are usually called the Ypt proteins in yeast and the Rab proteins in higher cells (reviewed in [i’] >, and appear to function in membrane traf-fc. Thus, in budding yeast the SEC4 and YPTZ gene products act at different stages of the secre- tory pathway. In mammalian cells, ditferent Rabs tend to be found on different membrane compartments, and ev- idence is accumulating that the Rab proteins are actively involved in membrane trafl?c in these cells, too (see [l] >. It appears that each Rab is attached to the cytoplasmic face of a particular class of transport vesicle, and plays a role in determining the target membrane with which the vesicle will fuse. For the initial attachment to the lipid bilayer, the Rab proteins, like was, require prenylation of their termi- nal cysteine residues. The problem was that this residue is found either as CC or as CXC, neither of which are substrates for the CAAX transferases, and yet both clearly become modified with geranylgeranyl groups; in addi tion, the CXC sequence acquires a methyl group. Thus, the hunt began for the Rab geranylgeranyl transferase. The Erst advance was the cloning of another yeast secre- toly gene, BET2, the product of which regulates mem- brane attachment of both Yptl and Sed proteins: it turned out to be a subunit of the yeast transferase [8]. Now Seabra et al. [2] have puriEed component A of Rab geranylgeranyl transferase from rat brain. The enzyme modiEes both CC and CXC termini. The latter, at least, is prenylated on both cysteines, and the simplest picture is that this single activity modiEes both cysteines on both sorts of terminus. However, more than one group has found wrinkles in this seemingly smooth stoory [d] . The enzyme has two separable components. Component B has two subunits, one of which is probably the homo- logue of the BET2 gene product, yet both are analogous to the two subunits of the other prenyl transferases. Com- ponent A, however, is new: a 95 kD protein, from which a partial amino-acid sequence has revealed a similarity to one known protein, as well as an apparent identity to another. The similarity is very sensible; the identity, at’ present, is not. The similar protein is called GDI, for GDP dissociation inhibitor. This factor was originally identified in studies of the genes Tab% (alias smgp25A). Its name does not convey what now appears to be its true function - re- cycling Rab proteins from the membrane into the cytosol [9]. This is an essential stage in the cyclic action of the Rab proteins (Fig. 1). Like its relative, Ras, Rab is thought to switch between an ‘active’ GTP-bound form and an ‘inactive’ GDP-bound form. Once a vesicle has found its target, a GAP activity inactivates the Rab protein, which,

@ Current Biology 1993, Vol 3 No 1 33

34 Current Biology 1993, Vol 3 No 1

Fig. 1. A comparison of the possible roles of GDI and component A. (a) GDI extracts GDP-bound prenylated Rab protein from a membrane, allowing it to be recycled to the vesicle membrane from which it came. GTP-bound Rab allows a vesicle to dock with its target membrane, and is then stimulated to hydrolyse CTP to GDP by a GAP activity. (b) Component A may present the substrates, newly-synthesized Rab and geranylgeranyl pyrophosphate, to the catalytic site on component B. Note that GDI is specific for the GDP-bound form of the Rab and can act repeatedly, whereas component A will recognize any form 121 and acts only once after synthesis.

by now, is in the membrane of the destination organelle. For Rab to be re-used, it must be returned to its original membrane; but the two geranylgemnyl groups constitute a ti anchor in the lipid bilayer, and the thioether link is chemically and biologically stable. GDI’s job is to extract the GDP-bound Rab protlein out of the membrane and back into the cytosol, acting as a sort of lipid ‘chaper- one’ for the hydrophobic isoprenyl groups. In this soluble form, the complex can then d8use back to the I&b pro- tein’s original membrane, where other proteins reinsert it in the bilayer and exchange GTP for GDP. It appears that a single GDI can act on most, or all, Rabs [lo]; hence it must recognize ‘rabness’ -- in other words, characteristics that identify this group within the was superfamily.

This is exactly the property required of component A Although the other prenyl transferases need little more than the tetrapeptide CAAX as substrate, the Rab trans- ferase will accept nothing less than an intact protein, and hence the additional subunit is the obvious car& date for fulfilling the function of substrate recognition. Equally obvious is the common interest of GDI and com- ponent A in geranylgeranyll groups. Indeed, the sequence similarity has been known longer than the existence of component A, because it was noticed first in its alter ego, the choroideraemia gene product [ 111.

Choroideraemia, also known as tapetochoroidal dystro- phy, is an inherited disease that results in slow degener- ation of the retina, ultimately lea$ng to blindness [12]. Genetic and molecular methods allowed mapping of the gene to the X chromosome and its subsequent identifi- cation: deletions, rearrangements or other alterations in expression of the gene result in the disease [ 131. What possible connection can there be between the disease, involving degeneration over a period of decades in spe- cialized tissues, and Rab prenylation, a necessary step in the action of members of an ubiquitous protein family, several of which are essential for viability in yeast?

What we do know is that, thankfully, the disease is not immediately fatal, suggesting that there is a family of com- ponent As. Rat brain, however, appears to contain only a single type of component A Even if there are other pro- teins working in parallel, why should the retina in particu- lar suffer and why does it take so long? Seabra et al. make the ingenious speculation that, as the rod outer segments are very active in membrane turnover, this process may be deficient in choroideraemia patients, ultimately lead- ing to cell death. The retinal cells, then, would require larger quantities of Rab proteins, and hence of compo- nent A, than most cells. The availability of cell lines from these patients, and the likely emergence of a component

3s A homologue in yeast, will allow a range of molecular and genetic investigations, but connecting those to the pathol- ogy of the disease may still be a formidable challenge. The discovery of the identity of component A is a sober- ing lesson for gene mappers. Cremers et al. [13], not- ing that defects in the activities of certain ubiquitous proteins, such as ornithine aminotransferase, result in symptoms similar to choroideraemia, wisely suggested that the choroideraemia gene product might also be a widespread protein. But, it is hard to imagine that the function of the choroidaemia gene product would ever have emerged simply from sequence gazing. Rumours of the imminent death of biochemistry, or its engulfment by more highly-publicized areas; of biology, would appear to be premature.

References 1. MARsH M, CUTLER D: Taking the Rabs off endocytosis. Curr

Biol 1992, 3:3@32. 2. SEABRA MC, BROWN MS, %&LIGHTER CA, SUDHOF TC, GOLDSTEIN

JL: Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideraemia gene product. Cell 1992, 7’0:104~1057.

3. KAMIYA Y, SUKURAI A, TAMURA S, TAKAHASHI N, TSUCHIYA E, ABE K, FUKUI S: Structure of rhodotaurucine A, a peptidyl fat- tor, inducing mating tube formation in Rbodosporid ium torahid&. Aeric Bio N&em 1979. 43:363-369.

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BOURNE H, SANIJEPZ DA, MCCORMICK F: The GTPme super- family: conserved structure and molecular mechanism. Nature 1991, 349:117-127. MAGEE T, NEWMAN C: The role of lipid anchors for small G proteins in membrane traft3cking. Trenak Cell Biol 1992, 2:31%323. Gout B, MCCAFFREY M: Small GTP-binding proteins and their role in transport. Curr Opin Cell Bioll991, 3:626633. Ross1 G, JIANG Y, NEWMAN A, FERRO-NOVICK S: Dependence of Yptl and Sec4 membrane attachment on Beta. Nature 1991, 351:15%161. ARAKI S, KAIEXJCHI K, SASAKI T, HATA Y, TAKAI Y: Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein. A401 Cell Biol 1991, 11:143t?-1447. SASAKI T, KAIBUCHI K, KABCENEIL AK, NOVICK PJ, TAKAI Y: A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibit) for smg p25A is active on yeast SEC4 protein. Mol Cell Biol 1991, 11:2903-2912. FOUR E, LEE RT, O’DONNELL JJ: Analysis of choroideraemia gene. Nature 1991, 351614. HECKENLIVEIEY JRI Retinitispigmentosa. New York: J.B. Lippincon Company; 1983. CREMERS FPM, VAN DER POL DJR, VAN KERKJIOFF LPM, WIERINGA B, ROPERS H-H: Cloning of a gene that is re arranged in patients with choroideraemia. Nature 1990, 347674-677.

4. HANCOCK JF, MAGEE-AI, CHILDS JE, MA&ALL CJ: AU ras pro- teins are polyisoprenylated but only some are palmi- John Armstrong, School of Biological Sciences, University toylated. Cell 1989, 571167’1177. of Sussex, Falrner, Brighton BNl 9QG, UK.

IN THE JUNE 1993 ISSUE OF CURRENT OPINION IN GENETICS AND DEVELOPMENT

Huntington Willard and Kay Davies will edit the following reviews on Mammalian Genetics:

Polygenic disease by J Bell Wilm’s tumour gene and function by N Hastie

Chromosome structure by H Willard and C Tyler Smith Rearrangements of chromosome 22 and human disease by P Scambler

Dystrophin and related genes by J Tin&y, K Davies and D Blake Hu:man gene map based on microsatellites by J Weissenbach

Advances in inherited metabolic disease by R McInnes Genetics of early mouse development by T Magnuson Integrating maps of chromosome 16 by G Sutherland

Trinucleotides and genome variation by T Caskey CMT duplication and candidate genes by P Pate1 Prader Willi/Angelman’s syndrome by R Nichols Integrating maps of chromosome 11 by G Evans

Aneuploidy and non-disjunction by T Hassold Mitochondrial diseases by A Schapira

Huntington’s disease by J Murray DNA repair genes by M Buchwald

Gene therapy by J Wilson