Structure and biological effects of lipid modifications on proteins Marie

Chow,

Channing

J. Der and Janice E. Buss

Massachusetts Institute of Technology, Cambridge, Massachusetts and La Jolla Cancer Research Foundation, La Jolla, California, USA Both the prevelance of lipid modifications of proteins and their importance for protein function and cellular localization have been widely observed. The advances made during the past year in defining the enzymology of lipid addition and in understanding the biological consequences of these modifications on protein function are discussed.

Current

Opinion

in Cell Biology

Introduction

1992, 4:629-636

alyze these modifications are being identified, has helped to establish that this previously obscure protein modification is a critical requirement for protein function in many essential cellular processes. With this new information, a reassessment of the similarities and differences between the different modifications is appropriate. Thus, we will attempt to identify general features or models that may be conceptually relevant. For more detailed discussions, readers are referred to several recent reviews [3-71.

the last decade, numerous experiments have demonstrated that proteins are modified post-translationally by fatty acids (myristate and palmitate), isoprenoids (famesol and geranylgeranol) and glycophospholipids (glycophosphatidylinositol, GPI). Estimates suggest that isoprenoid-modified proteins alone comprise 0.5-2% of all cellular proteins [ 1.1 and that, on a quantitative basis, l&50% of all proteins within mammalian cells may be modified by at least one of these lipid moieties [2]. Despite the prevelance of modified proteins, in many cases neither the enzymes responsible for the modifications nor the biological role(s) of the modification itself are known. The general roles that the lipid moiety plays in protein function and correct subcellular localization are now becoming understood. The consequent impact of these modifications is varied and they impinge on numerous apparently disparate biological processes, such as signal transduction and growth regulation, intracellular vesicular transport, cytoskeletal organization, membrane targeting and turnover, and replication strategies of pathogenic organisms. As many of the affected pathways are related to pathological disease states, attention has focused on lipid modification reactions as potential therapeutic targets. In the past year, rapid advances have been made in characterizing the enzymology and potential biological roles of these modifications. In particular, progress has been made in defining the catal@ mechanism for myristoyl addition and the biological contributions of the myristoyl moiety. Knowledge of the GPI structure and the ability to make protein chimeras with either GPI or transmembrane protein anchors have led to characterization of the GPI bio.synthetic pathway, the protein elements signaling GPI addition and the biological functions of GPI anchors. Finally, the explosive rate with which new prenylated proteins and the enzymes that cat-

Over

lipid

biosynthesis

and protein

modification

N-myristoyiation

Protein N-myristoylation involves co-translational attachment of myristate (Cl401 to the amino-terminal glycine residue by the soluble N-myristoyltransferase (NMT). Using gmthetic peptides, the substrate specificity for Sacchamn?qces eel-ezjisine NMT (to date, the only NMT purified to homogenity) has been extensively examined [6]. The studies demonstrate that the enzyme has distinct binding sites for the fatty acyl-CoA and peptide substrate. Myristate is donated from myristoyl-CoA and the enzyme’s unique acy-CoA specificity appears to be due to its ability to monitor the acyl-CoA length [8*]. An absolute requirement for the amino-terminal glycine residue is observed. No peptidase activity is associated with the NMT. Thus, N-myristoylation is dependent on proteolytic removal of the initiating methionine and exposure of the amino-terminal glycine. In addition, approximately seven amino acid residues adjacent to the amino-terminal glycine residue are also required for recognition by the cellular NMT. More recently, dissection of the catalytic mechanism has been possible with the development of heteroatom-substituted myristate and myristateCoA analogs (8*,9**]. The peptide substrate specificity

Abbreviations FPP-farnesyl pyrophosphate; FTase-farnesyl GGPP-geranylgeranyl pyrophosphate;

transferase; MARCKSmyristoylated

@

Current

CCTase-geranylgeranyltransferase; protein kinase C substrate;

Biology

Ltd

ISSN

0955-0674

CPI-glycophosphatidylinositol; NMT-N-myristoyltransferase.

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630

Membranes

appears to be affected by the physicochemical properties of amino acids distributed over the amino-terminal region of the protein and is dramatically influenced b) the bound acy-CoA substrate [8*]. In addition, the reaction proceeds via a sequential ordered Bi-Bi catalytic mechanism. That is, NMT binds to the myristoyi-CoA before binding to the peptide; catalysis occurs, generating CoA and the acyi-modified peptide; and, finally, CoA is released from the enzyme complex, followed by dissociation of the NMT and the myristoyi-peptide [p*]. As binding of the peptide and acyi substrates by the cellular NMT is highly cooperative, a given \iral or cellular peptide sequence is modified by different hetero-atom-substituted myristoyl analogs to different efficiencies. Thus, knowledge of the catalytic mechanism and substrate-recognition determinants of the cellular NMT allows development of anti-viral and anti-neopiltstic agents that may mod@ viral or oncogenic proteins and display therapeutic selectivity with minimal cellular toxicity [lO,ll*]. Although the majority of identified myristoyi-modified proteins are modified by mammalian NMTs, the substrate specificities and catalytic mechanism of these enzymes, which are often potential therapeutic targets, have been largely inferred from yeast NMT studies. Thus, the recent cloning and expression of human NMT presents a significant opportunity to characterize directly the enzymoiog) of a mammalian NMT [ 12**]. This study indicates that there are small but significant differences between the substrate specificities of human and yeast NMTs. Thus, further characterization of substrate specilicities for the human enzyme will be invaluable for future de\reiopment of human chemotherapeutic agents.

Prenylation

Protein prenyiation, the most recently identified class of lipid modification, involves the post-translational attachment of isoprenoids via a thioether linkage to cysteine residues at or near the carbox)li terminus [4,5]. ISOprenoid modification seems to be a stable irreversible addition, similar to myristoyiation. Two types of modifications have been identified: the addition of farnesyi or geranylgeranyi moieties. Farnesoi is a 15 carbon compound, which is composed of three isoprene groups and exists within the ceil as famesyi pyrophosphate (FPP), an intermediate in cholesterol bio.synthesis. The more abundant modification (SO-90%) occurs by addition of a geranyigeranyi moiety, a 20 carbon compound composed of four isoprene groups, from geranyigeranyi pyrophosphate (GGPP). GGPP is not an intermediate of cholesterol biosynthesis, and is presently associated only with protein prenylation in mammalian ceils [I*]. Prenyiation of CXXX proteins, (where C is cysteine and X is any amino acid), is accompanied by two additional, tightly linked modifications - proteoiytic removal of the terminal Xxx residues, followed by carboxymethylation of the now terminal cysteine residue. Although no signal motif has been defined, some prenyiated proteins (e.g. HRas) undergo further fatty acid modification by addition of palmitate (Cl6:O) via a thioester linkage to cysteine

residues upstream of the CXXX motif. The enzymes catalyzing the proteoi)zic processing, carboxTmethyiation and paimitoyiation reactions are beginning to be identified and characterized [ 13.1. However, they appear to be membrane-bound and dependent on the presence of the prenyi modification [5]. Within this past year, the number of identified sequence motifs that probably signal isoprenoid addition has gronn from one to six. Prenylated proteins terminating with the consensus carboxyi-terminal CX,X,X3 motif are modified by either fame.syl or geranyigeranyl moieties [ 1*,14=*,15**]. Residues at Xl or X7 affect the efficiency of the prenyiation reaction [ 14 *,16*,17*] The specificity of the reaction is determined largely by the last amino acid residue of the motif, Xj: this may be serine, methionine, cysteine. alanine or glutamine, mrhich signal addition of ;I farnesyi moiety, or ieucine or phenyiaianine, which signal addition of a geranyigeranyi moiev [ lt**.lS**,lb~]. Both c)steines of the Ras-related Rab proteins that terminate with either CC or CXC motifs are geran~lgeran!,i~modifed [l&(19,20*], Additional prenyiation motifs are thought to be represented by other Rab proteins that terminate with CCXX (Rab5 ), CCXXX (Rabl 1) or CCX (Rati) carboxyterminal sequences, although this has yet to be conlirmed [ 20*]. Given the possible permutations in prenyiation sequences and additional \ariation due to the associated modifications (painiito),iation, carbo~~methyiation and phospho~iation ). which occur to difierent extents, the population of an)’ modilied protein within the ceil ma! be quite heterogeneous nith respect to its lipid moditications.

l

Concomitant with the identilication of additional pren!.iation sequences, biochemical and genetic studies of the protein prenJ.1 transferases in mammalian and yeast ceils ha\re generated a picture of equal complexity. The tirst prenyi transferase to be identified and cloned, farnesyi transferase ( FTase 1, specifically modities Ras and nuclear lamin proteins (CXXX motif) with farne.syl groups [ 16*,21]. FTase is a c?Tosoiic enzyme that exists as an c$heterodimer. The P-subunit binds the peptide sub strate and the cc-subunit is believed to bind FPP. The individual subunits are apparently unstable kz 11irtoand the intact ap-complex is required for stable binding of FPP. The a- and b-subunits are the mammalian homologs of the yeast RAM2 and DPRl ‘RAIMl gene products, respectively [22,23-l. Yeast mutants defective for either gene product are impaired in FTase acti\lty A geranylgeranyitransferase ( GGTase), GGTaseI, has also been identified for the geranylgerany-modified CXXX motif proteins [ 13**.13**]. GGTasel is also a heterodimer. Although the GGTaseI P-subunit (homoiog of yeast Cdc-i3/Cail) is distinct from the FTase p-subunit, the GGTaseI a-subunit appears indistinguishable from and may be identical to the FTase a-subunit [ 15**,21]. If the a-subunit is involved in the binding of the prenyi pyrophosphate substrate (FPP or GGPP ), then selection of the lipid substrate depends on the p-subunit (GGTaseI or FTase) with lvhich the a-subunit interacts and raises the question as to how this selectivity is achieved. If the binding of the peptide and prenyi pyrophosphate substrates is highly cooperative, then, as in the case of N-myristoyitransferases, the

Effects

selectivity of the a-subunit may be achieved by its interactions with a specific P-subunit on its own, or with the peptide-bound P-subunit within the prenyl transferase complex. The Rab CC, CXC or CCXX motifs appear to be modified by prenyl transferase(s), such as GGTaseII, which are distinct from FTase or GGTase1 [ 14**,25]. The Rab prenyl transferases do not have the same a-subunit as the CXXXmodifying enzymes. Furthermore, these geranylgeranyl transferases may require recognition of the CC/CXC/CCXX motifs within the structural context of the protein (CJ Der, unpublished data). It is not yet known if distinct enzymes exist for each motif. However, the product of the yeast BET2IORF2gene is required for prenylation of CC-tenninating proteins [ 260). BET2 shows significant sequence homology with both DPRliRAMl and CDC43/CALl gene products, suggesting that it may be the P-subunit of CCmodifying GGTase. In addition, because both cysteines (CC and CXC motifs) are geranylgeranyl-modified, it is likely that each cysteine modification is specified by a different GGTase. Thus, there may be several GGTases with varying specificities within the cell. With identification of the Rab prenylation motifs, substantial progress in the isolation and characterization of these additional GGTase activities is anticipated.

Glycophospholipid

addition

The carboxyl termini of many functionally different cell surface proteins are modified with a GPI moiev. Although a variety of mature GPI structures has been observed, the core structure of the GPI moiety consists of phosphatidylinositoI (PI) linked to a tetrasaccharide (Nacetylgalactosamine-mannosel), which has a phosphoethanolamine attached to m&nose. The ethanolamine is attached via an amide bond to the a-carbo.xyl group of type 1 membrane proteins [ 3,271. The structure of the mature GPIs varies in the amount of a-mannose residues, the presence of an additional ethanolamine phosphate and/or palmitate modification, and the structure of the glycans that branch from the core glyco.syl residues. The PI lipid structures also vary. Although glycerol-based phospholipids in eukaryotic membranes usually contain a saturated fatty acid and an unsaturated fatty acid esterified at the Rl and R2 positions of the glycerol backbone, respectively, in several of the characterized GPI structures, saturated fatty acids appear to be esterified at both the Rl and the R2 positions. Apart from the GPI moieties .syrthesized in Tqpamsoma brucei, it is not known whether the acyl composition observed in mature GPI structures indicates that a specific PI moiety is used for synthesis of GPI structures, or merely reflects variations in the membrane composition observed for different eukaryotes. Two elements within the modified protein are required for GPI addition: an amino-terminal signal sequence, which enables transport into the lumen of the endoplasmic reticulum, and a GPI addition sequence. Although there is no unique consensus sequence for this last element, characterization of protein chimeras with GPI anchors or transmembrane domains has illustrated that the addition sequence is formed by a short hydrophobic do-

of lipid

modifications

on

proteins

Chow, Der, Buss 631

main (15-20 residues) 10-l 2 residues carboxyl-terminal to a pair of small residues (aspartic acid, asparagine, serine, glycine, alanine or cysteine) [ 28**,29]. Cleavage and GPI attachment occur between the small residue pair. Elucidation of the GPI structure has allowed the relevant biosynthetic precursors to be identified and the synthetic pathway for this lipid moiety to be dissected in cellfree .systems [30,31]. The variety of GPI structures observed in nature suggests that chemotherapeutic agents could be designed to interfere selectivelelywith certain GPI structures, and not others. Specifically, in 7: br-ucei, the dimyristoyl remodeling reactions of PI during GPI .synthesis may be effective therapeutic targets, as these parasites display selective toxicity to specific myristoyl analogues [ 32.1. The general effectiveness of this approach awaits further identification and characterization of the enzymes within this pathway. The transfer of labeled GPI anchors to endogenous polypeptide acceptors has recently been demonstrated in a cell-free trypanosome system [ 33**]. Similar progress is anticipated in the identification and characterization of enzymes directly responsible for the cleavage and GPI attachment in the same system.

General

comments

Structural studies have provided detailed analysis of each lipid modification and delineated important parameters of the modification pathways. Interestingly, each lipid attachment is frequently accompanied by additional protein modification, most notably proteolytic processing. For myristoylation and prenylation, proteolysis appears to occur via enzymes that are distinct from those involved in the lipid addition reaction. The cell biology of the modification reactions is still relatively poorly understood. Although the NMT and prenyl transferases are known to behave a$ soluble or peripheral membrane proteins during purification, whether the reactions occur in the cytoplasm or in association with the endoplasmic reticulum has yet to be established. In addition, it is not known whether the lipid precursors are generated within the same cellular compartment (and are thus readily available) or if they are transported from other cellular compartments. The interaction and regulation of these modification pathways with other pathways in lipid metabolism (lipid biosynthesis, fatty acid biosynthesis and oxidation, sterol and cholesterol metabolism) are still largely uncharacterized.

Functional

roles of lipid

modification

Early models proposed attractively that the function of the lipid modifications described above was to target proteins to cellular membrane compartments. Both the existence of soluble, non-membraneous, myristoylated and prenylated proteins and of an independent membrane signal sequence on GPI-anchored proteins clearly demonstrate that these modifications are not themselves membrane-targeting signals and that, for the myristoylated and prenylated proteins, membrane attachment is

632

Membranes

not solely due to the hydrophobic toyl or isoprenyl moieties.

nature.of

the myris-

N-myristoylation

The necessity of the myristoyl modification is most clearly demonstrated within viral systems, where mutants defective in myristoylation (i.e. my&ate minus) are non-viable [7]. The variety of effects obsenred when the myristoyl moiety is absent in different cellular and viral systems indicates that the underlying role(s) of the lipid modification for protein function appears complex. However, these effects may reflect the varying roles of the myristoyl protein within each of the biological systems rather than differences per se in my&ate functions. Although the UI~derlying biochemical mechanisms are % yet unidentified, myristate appears to mediate protein-protein interactions in each of the cellular and viral systems studied The resultant effects observed in each system upon perturbation or absence of the myristoyl moiety depend on whether the myristoyl mediated protein-protein interactions provide structural, kinetic or stabilizing functions, or a combination of these. In several systems, localization of the myristoyl protein to plasma membranes may be mediated by receptors. For example, a plasma membrane protein has been identified that binds the amino-terminal sequence of ppbO\‘-src and the myristoyl moiety is an essential component for ligand recognition and binding by this ppbO\‘-src receptor [ 341. Similarly, interaction of myristoylated protein kinase C substrate (MARCKS) protein with the plas~na membrane appears to be receptor-mediated [35*], For the heterotrimeric G proteins, myristoyl modification enables the G,, cl-subunit to form a high-alkit) complex with the p- and y-subunits on the membrane in the presence of GDP; m)~ristoylation of G,, a-subunits facilitates fonnation of the heterotrimer and localization of a-subunit to the plasma membrane [36**]. Thus, the membrane-associated G,, fi- and y-subunits act as receptors for the G, a-subunit. It still remains to be determined whether membrane localization of myristoylated proteins is generally receptor-mediated and whether the pp60~ src receptor is the protot)l>e of a family of myristoyl-dependent protein receptors. Differing roles appear to have been discovered for myristoylated viral proteins, many of which are structural proteins within the virus particle. Myristoyl modification is required both for membrane association of the Gag proteins and for assembly of retroviruses [37]. In poliovirus. the myristoyl moiety has a structural role within the assembled virus particle [38]. Myristoylation also increases the kinetics with which a pentamer subunit (an obligate assembly intermediate of the virion particle) is formed and is required during late stage virion maturation [ 39**] In other systems, virus particles are formed, but they are not infectious or they show reduced infectivity, suggesting that the myristoyl moiety may affect virion stability or may be required during virus entry [40, 41). Although these data are not inconsistent with myristoyl modification mediating viral protein interactions, the underlying

contributions of the myristoyl moiety may be multiple and are diffkult to dissect within the replication cycle of the virus. The early co-translational addition of my&ate to proteins provides an additional level of complexity that is not observed with the other lipid modifications; the modification is present both during .synthesis of the polypeptide chain and throughout the entire lifetime of the protein. Thus, the myristoyl modification may affect mukpie stages of protein structure and/or function, starting as early as the folding of the nascent protein chain. Although this may be relevant for cellular proteins, it is particularl) significant in assessing viral systems, as virus entry, virion assembly and overall virion stabiliv can be simultaneously affected [ 39**]. The outward manifestation of myristate absence on these pathways may be subtle, e.g. slower kinetics, or dramatic, e.g. virus death. Finally, the multi~ pie stages of myristoyl action provide multiple points of potential intemention by chemotherapeutic agents, some stages being more selective that others.

Prenylation

The best characterized prenylated proteins are the Ras proteins (CXXX motifs). The critical role of prenylation in protein function is demonstrated by studies in which mutant Ras proteins that do not undergo isoprenoid modification no longer associate with the plasma membrane and are non-transforming [ 51. Although the specilic contribution of each CXXX-signaled modifcation has not been established for normal Ras function, farnesylation alone is sufficient to promote efficient membrane association and transforming actkit) of oncogenic Ras proteins [ 1791. Similarly, in other systems, perturbing prenytation signilkantl!. alters the membrane association and biological activities of other prenylated proteins such as the G y-subunits, Rab proteins and nuclear lamins [-t,j]. Recently, prenylation of the Hepatitis delta virus large antigen has been reported [-+2**]. In this system, prenyl modification is required for the inclusion of the delta antigen into Hepatitis B surface antigen-containing \irus particles. Prenylation may direct localization of the delta antigen to membranes, thus promoting its incorporation into virus l~~-Wes. Alternativel~~,prenylation of large delta antigen rnq~ be required specifcall!~ for \irion formation and release. In this latter scenario, the prcnyl moiet)., like the myristoll moietj~, may be influencing the tertiary structure of the delta antigen, rather than fulfilling a membrane-targeting role. Such stuctural intluences ma)’ be particularly impomant for small proteins such as the Lmil!, of Ras and Kas-related proteins. The ekcts of prenylation on protein structure are unexplored. The effects of the prenyl moditication on the ability of a protein to interact or associate with membranes is independent from the effects of that moiety in targeting the protein to specilic subcellular membrane compartments. Both Ras and lamin B undergo the same CXXXsignaied modifications, but & is targeted to the plasma membrane while lamin B is associated with the nuclear envelope. In addition, all Rab proteins, which

Effects

function in regulating vesicular transport, are apparently similarly geranylgeranylated, yet each associates with a distinct subcellular membrane compartment [43]. Furthermore, a significant fraction of prenylated proteins is present in the cytosol. Thus, although prenylation is critical to trigger membrane association, additional information is required for the specific subcellular targeting of prenylated proteins [ 44**]. The nature of the targeting information is complex. For CXXX-modified proteins, additional modifications (e.g. palmitate addition) or protein sequences (e.g. lysine-arginine rich domains) upstream of the prenyl modification appear to function as extra signals to promote the specific targeting of these proteins to their correct intracellular location [45**]. The exclusive plasma membrdne location of palmitoylated proteins suggests the presence of receptors that may recognize both the acyl and isoprenyl moieties. In addition, the vety labile, apparently reversible, nature of the palmitoy1 modification is unique and contributes to the microheterogeneity in lipid modifications observed so far. The reversability of the palmitoyl modification in conjunction with a putative palmitoyl-dependent receptor could provide an alternate mechanism for dynamic interactions with other proteins and/or substrates at the plasma membrane. For the Rab proteins (CC / CXC / CCXX / CCXXX / CCX motifs), the complexity of the prenylation signal sequences and their modification patterns may reflect their complex roles in regulating unidirectional vesicular transport processes between discrete compartments of the endocytic and exocytic pathways [43]. Although it remains to be determined, the intracellular localization of the different prenylated proteins may be mediated (as obsenled for myristoyl-modified proteins) by interactions with prenylation-dependent receptors. Alternatively, different hydrophobic characteristics are conferred upon the protein by the farnesyl and geranylgeranyl moieties; these differences could be further augmented by the presence of two geranylgeranyl moieties, palmitoyl addition or carbox3~lethylation. Given that the lipid compositions may be very distinct within different subcellular membrane compartments, the apparent targeting could reflect the affinity of these prenylated proteins for membranes with differing physical properties. Conversely, one could imagine that the insertion of significant numbers of prenyl or palmitate moieties (as might be envisioned with the Rab proteins) might affect the physical/chemical properties of the membranes themselves in the different vesicular or endocytic compartments. Within the cell, each prenylated protein is specifically modified with either farnesyl or geranylgeranyl moieties. Studies with was indicate that Ras-transforming activity can be promoted by the presence of either famesyl or geranylgeranyl moieties [45**,46**] or even N-myristoy1 modification [ 51. However, potent growth inhibition of NIH3T3 cells is observed when normal Ras protein is geranylgeranyl-modified [46*-l. This is in contrast to the normal growth observed after farnesyl modification. These data indicate that modification with a specific isoprenoid is important for protein function and that each

of lipid

modifications

on

proteins

Chow,

Der, Buss

lipid modification has a specific role for protein function that cannot be provided by another lipid. In addition, the heterogeneity in lipid modifications present on any given protein may provide subtle differences in protein function and protein-protein interactions that may be highly significant in regulating and modulating cellular processes. Understanding the precise contributions of prenylation to protein function remains an important goal for future studies.

CPI

anchors

The GPI anchor can be considered as an alternative to the transmembrane peptide domain for ensuring membrane association of type 1 membrane proteins. However, the apparent functions of the GPI anchor have become increasingly complex with additional study. Although aU GPI-linked proteins are expressed on the cell surface, in polarized cells they are specifically sorted to the apical surfaces [47+]. Whether this localization is due to a default pathway or to the presence of specific receptors is unclear. In addition, the concentration of GPI-anchored proteins in clathrin-coated pits is substantially lower than other transmembrane receptor proteins, and the rate of clathrin-mediated endocytosis for these proteins is very slow compared with that of other intrinsic membrane proteins. Thus, the GPI anchor appears to increase the half-life of proteins on the cell surface [ 501. Other data indicate that the protein distribution in the membrane is heterogeneous over the cell surface. Transmembrane proteins appear to be concentrated within microdomains, whereas GPI-anchored proteins seem to move freely across these domain boundaries [51]. Conversely, the GPI-anchored folate receptor appears to cluster in numerous membrane invaginations or caveolae; this clustering is dependent on cholesterol [ 52,531, Given the great diversity of lipids found within a membrane, the segregation or distribution of different GPI-anchored proteins may be highly dependent on the interactions between the lipid moiety of the GPI anchor and potential membrane microdomains formed by slight differences in the distribution of lipid types and cholesterol. Thus, one could hypothesize that the biochemical microenvironments formed by the clustering and organization within membranes of these potential transmembrane and GPI protein microdomains (coupled with the apical sorting observed in polarized cells) influence the kinetics and regulation of the many biochemical or physiological processes that occur at the ceU surface. Finally, an attractive model remains that GPI anchors allow the surface expression of modified proteins to be modulated by cellular or serum phospholipases. Although phospholipase release of GPI proteins from membranes is observed in l&-o, few examples have been reported in ziljo. Thus, the observation that the pancreatic granule membrane protein, GP-2, appears to be secreted from the resting unstimulated pancreas by GPI cleavage, provides an example of a connection between the release of the GPI-anchored protein and the physiological state of the tissue [54*1.

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Membranes

Conclusion

.

Knowledge of how proteins are modified by lipids has rapidly evolved over the past few years from being a set of interesting but apparently esoteric reactions into a body of information with significant impact on cell biology and physiology. Although the presence of these modifications is critical for protein function, the exact contributions made by the lipid itself are unclear. The effects of the lipid modification on protein structure are virtually unexplored. The localization of modified proteins to specific subcellular compartments suggests the presence of receptors, whose ligand recognition is dependent on lipid modification, although these have yet to be identified. In addition, the diversity of lipid structures and compositions within different membrane compartments may strongly influence subcellular localization, structures of the modified proteins and ultimately protein function. Similarly, the lipids contributed by such modified proteins may significantly effect the structure and physiology of different subcellular membranes. GiLTenthe unexpected knowledge obtained to date, studies on the underlying mechanisms and effects of these lipid modification promise to provide additional unexpected insights at the interfaces between protein structure and function, membrane biochemistry and cell physiology.

References

and recommended

Papers of panicular interest, publishcil view, have been highlighted as. . of special interest .. of outstanding interest

reading

nithin

the annual

peritxl

of re

Triple Bonds and/or an Aromatic Residue. .I Rio/ Chenr 1991, 66:8835-8855. Ilrter.ltom-suhstirt~ myristoyl analogs are synthesized to characterize the suhsmw specificin of the yeast NMT for the acyl moie~. In addition to detining ke) elements for ac?l substrate recognition by the NMT. chatztcterlzation of the NMT cnzymolo~ using these types of analogs is important because of their potential use as chemother~prutic agents. RI~I)NICK DA, RICWHFRTER CA. ROCQ~W WJ, LENNON PJ, GErt&a DP. Go~ws Jl: Kinetic and Structural Evidence for a Sequential Ordered bi bi Mechanism of Catalysis by Saccharom)res cerevisine Myristoyl-CoA-Protein N.myristoyltransferace. ,I Rio1 Chon 1991. 669732-9739. The catalytic mechanism of yrr!st NMT is studied using myristoyl-CoA and a non-hydrolyzable analog of myristoyl-CoA. The data provide both a detailed description of the reaction mechanism for this enzyme and an rs1~erinient:tl hasis for the development I,f anti-twoplastic and antiviral ther.tpeutic agents nith greater selectiviv toward [his lipid moditication.

9. ..

Jolixsos DR. Cos AD. SOISKI PA, DE\‘al,ti B, ADMIS SP. Ixi!.!c;R~‘m% Ri\l. HI:I‘CKERO~II RO. Bt’ss JE. GORFXIN JI: Functional Analysis of Protein N-myristoylation: Metabolic Labeling Studies Using Three Oxygen-substituted Analogs of Myristic Acid and Cultured Mammalian Cells Provide Evidence for Protein-sequence-specific Incorporation and Analog-specific Redistribution. Proc ;%I/ Acad Sci 1 ‘S.4 1990. 87:X51 l-8515, BRYa&-l‘ hlL. R.\l-SeR L. D~‘RONIO RJ, ld%loRE NS, DE\‘.w,u B. AI)~L\I~ SP. axttxls JI: Incorporation of 1 .methoxydodecanoate into the Human Immunodeficiency Virus 1 gag Polyprotein Precursor Inhibits Its Proteolytic Processing and Virus Production in a Chronically Infected Human Lymphoid CelI Line. /‘tw ~Vcrll Acczd Sci (‘.SA 1991, 88:2055-2059. Describes the mechanism hy which a heteroatom-sul,stiturrd myristoyl analog inhihith human immunodrlicienc~ litus replication. Incorpora tion of this analog causes redistribution of the Pr55gag proteins from the pl&sma mrmbmne to c)w)solic fractions and inhibits late stages of \iruS assembly. 12. ..

1. .

E~s1-w WW. b:\+tt D. USING LM. BRI‘ENGER E. RUING IIC: Quantitation of Prenyl Cysteines by a Selective Cleavage Reaction. Ptw N&l Acrid Sci I’S,4 1991. 88:966+9670. This study establishes the relati\re abundance of the two known isoprenoids awx%Ated nith proteins in mammalian tissues and ytwt. fungal, algal, plant and inxct ceils. Ger.ln!lgerJnykTsteine is predomi nant over farnesylcysteine and the prenylc?stcine. protein content ratio indicates that isoprenylation is a common modilication of mammalian proteins.

D~UK>SIO (1. Rwn SI. Golux~~ 11: Mutations of Human Myrlstoyl-CoA-Protein N-myr&toyltransferase Cause Temperature-sensitive Myristic Acid Auxotrophy in Saccharornyces cererhiue. ./ BIO/ Clwnr 199 1. 89:-t I294 133. The cloning and characterization of the human KhlT by complementation in yeast is reporwd. This study is important because it prwides the tirst detailed characterization of a mammalian NMT.

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MAGEI: AI: Lipid Modification of Proteins and Its Relevance to Protein Targeting. .I Cell Sci 1990, 97:581-581.

Avwj’ XlN. WSG DS, Rts!i J: Endoproteolytic Processing of a Farnesylated Peptide In Vitro. PKK ~Vctfl .Iccrrl Sci l/S,4 1992, 89:-16134617. This report identifies a menlbrJlle-bound endoprotrase. Its acti!iv is dependent upOn a prenylated Substrate. Based on its insensitivity to a panel of protnse inhibitors. it appearh to &tine a new class of prott3.WS.

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M.UTESE WAN: Posttranslational Modification of Proteins b) Isoprenoids in Mammalian Cells. f%.SE~J 1990. 4:331+3328. 5.

KATO K. DER CJ. Btlss JE: that Control the Biological Cnncer Biol 1992, in press.

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TOWIIR DA, GORDON JI. AD.&hlS SP. GUSER L: Tbe Biology and Enzymology of Eukaryotic Protein Acylation. &UU Ret, Biochew~ 1988, 57:6’+99.

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CHOW M. 1hlosctwoN: Myristoylation Lipid Mod@carion o/ Proleim Edited Press, in press.

8. .

ffiSHORE NS, LLI T. WOU LJ, KATOH 4 RL’IXICK DA, hlEtIT;l PP. DEVADAS B, HtIHN M, A’nvoa~ Jl- Arxvs SP, ET a: The Substrate Specificity of Saccharomyces cerevisiue MyristoylCoA-Protein N-myristoyltransferase. Analysis of Myristic Acid Analogs Containing Oxygen, Sulfur, Double Bonds,

of Viral Proteins. In by Schlesinger M. CRC

13. .

Meows SL, SCHAI~ER MD. Mosw SD. RAW\ E. 0’H.w MB. G*KY \%I, h&twu.t. MS, Pt~htw\w DI, Gtws JB: Sequence Dependence of Protein Isoprenylation. ./ Rio/ Chen~ 1991, 66:1+603-li610. This study tdmtifirs in bovine tissue and yeast two distinct protein prenyl trJnsferfie acthities that m&i@ CXXX terminating peptides Rith either fames?1 (FTase) or geranylgeranyl (GGTasel) moieties and a third activity (GGTaseII). nhich geranylgemnylates the yeast ll-‘Tl protein (CC motif). The inahiliv of GGTaselI to prenyktte YPTl carboi?l-temiinal peptides distinguishes the sequence requirements for modilication by GGTaselI from the simple tetrapeptide requirements of CXXX.modifiing activities. F&.AM.A MC. RIISS Y, CAWY PJ, BROU?: IMS. GolnsT~% JI; Protein Farnesyltransferase and Geranylgeranyltransferase Share a Common ‘1 Subunit. Cell 1991, 65:nthesis and addition of GPI anchors. The authors describe how GPI can be transfered from its precursor molecules to endogenous polypeptide acceptors in a trypanosome cell.free system. This will enable identification of the enzymes directly involved in GPI anchor ad&ion. 33. ..

3-l.

&s~ MD, LING H: Identification of a 3 K Plasma Membrane Protein that Binds to the Myristylated Amino-terminal Sequence of P~O~‘-~~~. Nuiurc 1990. 346:8+86.

TIIELU hl. Rosw A, N.uw‘; AC, ADERU! A: Regulation by Phosphorylation of Reversible Association of a Myristoy lated Protein Kinase C Substrate with the Plasma Membrane. A’&rre 1991, 35 1:32&322. Describes how the interaction of the MARCKS protein nith the plasma membrane appt-.trs to he mediated by the phosphorylation state of the protein Its localization within specific regions of the pI%mJ membrane suggests that this interaction is receptor-mediated.

35. .

LINIXR ME. P&WC I-H. D~IRONIO RJ. GORDON JI, STER~WIS PC, GII~LW AG: Lipid Modifications of G Protein Subunits. Myristoylation of G, Increases Its Affinity for py. / Biol Cbem 1991, 66:-165-++659. Several biochemical asays were developed to investigate the functional role of myristoyl modification of the G,, a-subunit. These data show that the myristoylation increases the affinity of G,, a for the p- and y.subunits

36. ..

635

636

Membranes and that the localization of G, a to the plasma membwe is due to this &r&y. Although the underlying mechanism is unknown, these studies suggest that the myristoyl moiety may alfect the structure of the G,, a and that the structures of the modified and unmodified G,, a-subunits are not the same. 37.

SCHLILIZ AM, REW A: Unmyristylated Leukemia Virus Pr65SrrS is Excluded and Maturation Events. J Vim/ 1989,

Moloney from Virus 63:237C-2373.

Murine Assembl)

38.

CHOW M, NEWMAN JFE. FILMAN D, HCGLI! DM. ROUWDS DJ. BROWN F: Myristylation of Picomavirus Capsid Protein VP4 and Its Structural Significance. N&we 1987, 3 7:482-+86.

MOSCUFO N, CHOW M: Myristoylation is Important at Multi39. .. ple Stages in Poliovirus Assembly. ./ I’iral1991. 65:2312-2380. The functions of the myristoyl modification during poliovirus replica tion are examined using site-specilic tints mutants that dew-regulate myristoylation levels within the cell. Myristoylation appears to act a1 multiple stages of virus replication, during both virus assembly and entry 40.

MACW DR, BR~ISS V, GANEV D: Myristylation atitis B Virus Envelope Protein is Essential but not for Virus Assembly. \‘iro/og)t 1991,

of a Duck Hepfor Infecthit) 181:353-363.

41.

KRA~I~~W~CZ N, SIXE~IU CH. STUART-SMITH J. JONES MD, ~‘Au~u:I~ S. GRIFFIN BE: Myristylated Polyomavirus VP : Role in the Life Cycle of the Virus. ./ l’irol 1990. 64:441+-&O.

42. ..

GLESN JS, VIJA~ON JA. HA\IX CM. \Y:HI~-E JM. ldentilication of a Prenylation Site in Delta Virus Large Antigen. Scimw 1992. 56:1331-1333. This repon demonstrates that a Hepatitis delta virus protein is prenyl modified. In addition, it shone that prenyl moditication is required f(>r inclusion of this protein into \irion particles. This is the lirat report of a pren!l modificarion on viral proteins. Although the prvnyl m(Klihcation is hypothesized IO prwide a mcmhranr-targeting signal, its rc quirment for Lit-ion formation suggests that the m(xlitication may also have a structural role. 43.

BALCH WE. Low Molecular GTP-binding Proteins (LMGPs) Involved in Vesicular Transport: Binary Switches or Biological Transducers? Trwd~ Riochw Sci 199Q, 15~169-1’2.

CNAVIUER P. GOR\‘EL J-l’. .STEIZEH E. ~IMC)NS ti. GRI’INWRG J. ZERIAL M: Hypervariable C-terminal Domain of Rab Proteins Acts as a Targeting Signal. Natclrrrre 1991. 353:‘6+“2. This stu+ demonstntes that the carho~l.terminal serluences of Rahi and Rab7 proreins are required to complemenr the geranylgerdnylatmn modifications for spew-ific targeting to distinct zuhcellular membranes of the endtx)dc pathnay.

lation a\idiv

protein is shown both intracellular localization.

to increase

membrane-hindtng

46.

Cos AD. HISAKA MM, BLISS JE. DER CJ: Specific lsoprenoid Modification is Required for Function of Normal, but not Oncogenic, Ras Protein. J/o/ Cell Rio/ 1992, 1 :XL%-2615. This study prwides evidence that famesylation and geranylgerdnylation may have unique roles that are not interchangeable for protein function. Geranylgeranyl~modilied normal Ras proteins appeared to be growh inhibitog \vhen expressed in rodent fihroblast cells. ..

~12i.wr MP. ~,AIL.LS w, GIIBERT T, llAN731. D. ROIXUGI‘EZ~BOIU~ E: Vectoral Apical Delivery and Slow Endocytosis of a Glycolipid-anchored Fusion Protein in Transfected MDCK Cells. Prrx’ N&l Acad Sci I’SA 1990, 87:7419-‘423. -18.

Au N. E\‘;wh WH: Priority Targeting of Glycosylphosphatidylinositol.anchored Proteins to the Bile-canalicular (Apical) Plasma Membrane of Hepatocytes: Involvement of ‘Late’ Endosomes. /3iochm J 19W, 71:193%199.

19.

Darn CC. PAHTOS RG, SlXlo~s Glypiated Proteins in Hippocampal 349:1%+161.

i0.

~,5XIANSh~ I’. FaTl%W SII. (;ORICAS B, MEY.UI S. RO>SI~HO R. TART.+KOFF AM Dynamics and Longevity of the Glycolipidanchored Membrane Protein. Thy- 1. ./ (7ca// Rio/ 1990. 110:15’i-Ii31

iI

Erwis hl. S’I’HOYXOW~ I, Differences Between the Lateral Organization of Conventional and Inositol Phospholipidanchored Membrane Proteins. A Further Delinition of Micrometer Scale Membrane Domains ./ O,N Hiol 1991. 11 ~11~3~1150.

i2.

ROTIII~IXEK K. I’hTEtiON H. !V&L5H.UI. CJ. A CAAX or a CAAL Motif and a Second Signal are Sufficient for Plasma Membrane Targeting of Ras Proteins. El/HO ,I 1991, 10:-1033-l039. This repon is another in a series of outstanding studies on the role of I& cdrho?+enninal sequences and post-translational modilicationb in plasma membrane association and transfonnmg acti\-iv. This stud! demonstmtes that carhoxyl-terminal sequences directly upstream of the H-Ras and K.RastB CSXX monf are suticient to target a hrterologous. c)?osolic protein to the plasma membrane. Additionally, geranylgeran!

of K.RaslB and alter

K:

Polarized Neurons.

Sorting of Nnlfor 1991.

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