lsoprenoids

14. Sc;ihr;i, hl. C., liciss, Y.,Case,, 1’. J., Ijrowm, hl. S. & (;oldstein, J. 1.. ( 1 0 O l ) Cell (C;imbridge. Mass.) 65.

420- 4 34 15. liciss. I,..Str;idlcy. S.J.. (;irrasch. I,. hl.? Brown. M. S. 8r (;oldstein, J. I,. (1001) I’roc. Natl. Acad. Sri. 1i.S.A. 88. iZL-73f)

10. Yokoyarn;i, K., (;ood\vin. (;. W..(;hornashchi, I;. (;loinset, 1. A. 8r ( k l b , M. 11. (1001) I’roc. Natl. Acad. Sci. I1S.A. 88, 5.302-5300

497

Iicccived 1X I )rcembcr 100 1

Lipid modifications and function of the ras superfamily of proteins A. I. Magee,* C. M. H. Newman,* T. Giannakouros,* J. F. Hancock,t E. Fawell$ and J. Armstrong$ *National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 I AA, U.K., tAcademic Department of Haematology, Royal Free Hospital School of Medicine, Pond Street, Hampstead NW3 2QG U.K.,$Membrane Molecular Biology Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, U.K.

Introduction r .

I he rus superfamily of proteins consists of over 30 members \vhic*h are all low-molecul;ir-iiiass (20-30 kl )a) (3I’-biiiding proteins I 1 I. These can be

divided on the basis of over;ill sequence homology into three subfamilies: rus-related, involved in differentiation and gro\vth control; rho-related, some members regulating the cytoskeleton; rub-related. controlling intracellular niembrane trafficking. All these proteins function via a cycle of ( X I ’ binding and hydrolysis. In the (;I )I’-bouiid state they ;ire in an ‘inactive’ conformation. 1Inder the influence of a v;iriety of nucleotide exchmge fictors, the (;I )I’ is exchanged for (;Tl’ resulting in a conformational s\vitch to the ‘iictivr’ state, in \vhich form the protein can effect its function. The protein is turned off by ;in intrinsic (Jl’;ise activity \vhich hydrolyses the (3’1’ t o (;I )I’. often under thr influence of stirnulatory proteins such ;is rus-(;AI’ and NP-1. Although the roles of the rus-supert;imily members are diverse, most of them must be associated \vith the cytopl;ismic face of specific cellular membranes in order t o function. 7’;irgeting t o these specialized sites is achieved by co-operation between at least t t v o distinct regions of the proteins. The first is the extreme C-terminus \\here a series of lipid modifications increases the hydrophobicity o f the proteins and thus their membr;iiie binding affinity. Secondly, ii region of protein sequence -just upstream of the Cterminus (the hypervariable domain) app;irently encodes the determinants of targeting to the correct destination. presumably through interaction with membrane lipid head groups and/or membrane proteins.

ras proteins I’~ilmitoylation of one or more cysteine residues in

N- and H-rus proteins had been known for several years, but it was not until 1080 that the full process-

ing path\vay was elucidated. ‘I’he kej. to this was the recognition that the C-termin;il ‘CAAX box’ ( C ) cysteine; A. aliphatic amino acid; X, any amino acid) was the site of ii series of modifications similar t o those occurring o n ;i number of secreted fungal mating factors 12-4I. ‘I’hese involve (i) prenylation of the cysteine with the ( ‘ , i steroid precursor farnesol in thioether linkage; (ii) proteolytic renioval of AAX; (iii) carboxyl-methylation of the a-carboxyl group of the cysteine residue. In the case of the N-, I I- and K(A)-rus proteins these modifications are fi)llotved by palmitoylation of one o r two ne;irby upstream cysteine residues in the hypervariable domain. In contrast, the K(H)-ras protein lacks palmitoylation sites but instead has a polybasic region kvhich co-operates with the C‘AAX modifications t o specify plasma membrane binding. Kemoval of the palmitoylation sites o r the polybasic region results in normally CAAX-modified proteins which are nevertheless essentially cytosolic 15 1. Interestingly, the palmitate moieties of the rus proteins turn over rapidly in 7i710 (t,,? 20 min) suggesting a possible ~nechanisrnfor regulation of iictivity by modulation of membrane binding [ 6 1. All of the C‘AAX modifications can be reconstituted in vitro [7]. Farnesylation occurs in an unsupplemented reticulocyte lysate, demonstrating the presence of soluble prenyldiphosphate synthetases and protein: farnesyltransferase. Proteolysis and methylation, however, occur only after the addition of microsomal membranes and Sadenosylmethionine. All three of the modifications are required for full manifestation of membranebinding activity, and this is specific since it occurs primarily to added plasma membrane-enriched fractions rather than to the endoplasmic reticulumenriched microsomal membranes used as a source of protease and methyltransferase. Recently it has become clear that two types of CAAX box exist.

I992

Biochemical Society Transactions

498

Where X=Ser, Met. Cys, or (;In farnesol is added to the cysteine, whereas X = I,eu or I’he results in the addition of the C,,, isoprenoid geranylgeraniol by a geranylgeranyltransferase. ‘I’he subsequent rewtions of proteolysis and methylation appear to be the same. Indeed, recent evidence suggests that a single methyltr;insferase can niethylate both farnesylated and geranylgeranylated cysteines equally \\ell [ 8,0 1.

Fig. I Proposed

C-terminally

modified

-

Pal

H-ras

-Cys

I

Pal

Far

I

I

cys---cys

‘OCH,

GG

I

SpYPT1

.cys.-

cys

GG

Pal?

I

I

O ‘H

cys -cys

SpYPT3

\ O H

3

Volume 20

of

Abbreviations Far, farnesyl. GG. get anylget anyl. Pal, palmitate

rob proteins The rub subfamily contains many members with alternative C-terminal motifs o f the type XCC or CXC. We have been analysing the processing of members of this subfamily from the fission yeast Schizosucchuromyces pombe namely Y1’7’1, 3 and 5. I his organism has the advantage that its genetics is easily manipulated, giving the potential for gene replacement t o study function. The 17’7’1, 3 and 5 genes are all essential in S. pombe. In addition, n e have recently stioun that S. pombe, unlike Succhuromyces cerevisiue. \+ill efficiently take up and incorporate label from exogenous [ ’1 I Iriievalonic acid into proteins in the form o f farnesol arid geranylgeraniol I101. On the other hand. the usual inhibitors of hydroxymethylgliit~~ryl-CoA(I Ihl(;-Coh) reduct;we are ineffective in this organism. I lsing the same methodology as previously applied to the rus proteins 12, 1 1 I u e have studied the processing of these three YI’T proteins by translation in edro and after subcloning their genes into a mammalian expression vector folloued by transient expression in COS cells (C. Newman. 7’. Giannakouros, J. I Iancock, E. l;awell, J. Armstrong & A. 1. Magee, unpublished uork). Our results are summarized in Fig. 1. with the I I-rus protein for comparison. W e find all three proteins to be geranylgeranylated while only Y I”1’S is carboxylmethylated. Site-directed mutagenic analysis of the YP‘I’S protein (CXC) shows that both cysteines can be prenylated, and that there is no requirement for them to be modified in a specific order. Methytation only occurs on proteins which have a C-terminal prenylated cysteine (CXC or SXC, but not CXS) consistent with the known specificity of carboxylmethyltransferase [X, 01. Ilowever, the YP‘I’S protein is not metbylated in vitro under conditions where rus proteins are, suggesting the possibility of a second methyltransferase or a prior modification. Similar results have been obtained by studying the modifications of the YPT proteins endogenously expressed in S pombe. ‘I’he fact that YPTl and 3 proteins (XCC) are not methylated could b e interpreted to mean that

structures

5. pornbe YPT proteins compared with H-ros protein

GG SpYPT5

-

I

cys

GG

I

- x -cys, OCH,

they may be singly prenylated on the upstream cysteine residue only. and thus would not form a substrate for a Inettiyltransferiise. Alternatively, double prenylation of two adjacent cysteines might inhibit the activity of the methylating enzyme. ‘I’he resolution of these possibilities will await mutagenic and protein chemical analysis. We;ik. but apparently specific. incorporation o f I ’I I IpaImitate in thioester linkage into the Y 1’7.3 protein only has also been observed, but its signific;uice is iis yet unclear. Recent data suggest that the S. cerewkiue bet-2 gene product is a coniponent (probably ;I Bsubunit) of a geranylgeranyltraIisfer~isespecific for proteins of the XCC type 12 I. CXC-terminated proteins have not yet been reported in S. cerevisiue. but a geranylgeranyltransferase activity, active on proteins with a CXC‘ terminal motif has been detected in mammalian tissues I 13 I.

Function of protein-bound prenoids ‘I’he farnesyl moiety is of comparable hydrophobicity to palmitate and is insufficient on its own to mediate strong membrane binding 151. It is clear that this requires the co-operation of a further lipid modification (palmitoylation) or a polybasic region which may interact with the head-groups of negatively charged phospholipids such as phosphatidylserine, which is enriched in the plasma membrane. Thus de-palmitoylation or neutralization of positive charges, e.g. by phosphorylation, could allow rever-

lsoprenoids

sible association with membranes, which has been proposed to be involved in the cyclical functioning of some ras-related proteins. 1 he geranylgeranyl group. hom ever, is considerably more hydrophobic than farnesol and mediates tighter rnembrane binding [ 15 1. I n the case of rub 3 A [ 161 or Y P T S where a double geriinylgeranyl modification is present it is difficult to imagine how the protein could dissociate from membranes. This could be achieved by interaction Lvith a C-terminal binding protein such as smp~25A/rab3A-(;I)l which \vould cover up the hydrophobically modified site. resulting in forniation of ;I soluble complex [ 17 l. Although the C-terminal modifications of rob proteins are necessary for their membrane binding and specific targeting, they are cle;ii-Iy not sufficient. Intuitively it seems unlikely that enough specificity could be generated by these modifications, and upstream hypervariable sequences have also been s h o u n t o be essential 11x1. v 7

Conclusions The discovery of differential C-terminal modifications of rus proteins (farnesylation) and other niembers of the ras superfamily (geranylgeranylation) leaves open the possibility that specific farnesyltransferase inhibitors can be developed u hich will allow selective inhibition of ras function with minimal side-effects. One complication of this approach is that nuclear lamins, which are modified in an identic;il way to ras proteins, mill also be affected, as may other as yet unidentified farnesyIated proteins. Nevertheless, the further study of protein prenylation and associated modifications may provide spin-offs m it11 implications for the control of human disease, as well as further enhancing our understanding of the fundamental biochemistry and cell biology of eukaryotic organisrns.

I . I)ownw;ird, J. ( I W O ) Trends Iliochem. Sci. 15, 473-477 2. llancock, J. I;., Magee, A. I.. Childs, J. R. & Marshdl, C. J. (1 080) Cell (Cambridge, Mass.) 57, 1 167- 1 177 3. Schafer, W. K., Kim, K., Sterne, K.,‘I’horner, J.>Kim, S.-lI, & Kine, J. (10x0) Science 245, 370-384 4. ( h e y , 1’. J., Solski, 1’. A,, I k r , c‘. J. & Huss. J. E. (1080) I’roc. Natl. Acad. Sci. I:.S.A. 86, X323-8327 .5. I lancock. J. F.,I’atcrson, I I. & Marshall, C. J. (1000) Cell (Chinbridge, Mass.) 63, 133-1 30 0. Magee, 12. I., Grtierrcz, I,.? McKay, I. A,, Marshall, C.J. & Hall, A. (1087) 13MRO J. 6. 3353-3357 7 . I lancock, J. I:., Cad\vallader, I