Bio~himica et Biophysica Acta, 988 (1989) 411-426 41 l Elsevier BBAREV 85359 Fatty acylation of proteins Michael F.G. Schmidt Kmvmt Uniuo'sity. Facul~" of Medicine. Kuwai! (Arabian Gull) (Rec~ved 30 November 1988) Contents I. Introduction .............................................................. 411 II. Ester-type palmitoylation ..................................................... 412 A. Id~tif,cation of pahmtoy~tgd pm~chu of viral and c~llular origin ..................... 412 B. The scyl linkage s~tes in pa.lmitoylated proteins ................................... 414 C. Biochemistry of pslm/toylafion .............................................. 415 i. Location of pahmitoyhting activity ......................................... 415 7,. Dynamic nature of palmitoyladon .......................................... 416 3. Towards 0~e purification of a protein fatty acyluamferase (PAT) .................... 416 4. Sp~ficlty of lipid substmtes for ~-|ypc fauy acylation of proteins ................. 417 IlL N.terminal m)0ristoy~ .................................................... 417 A. ldentif~catioa of myristoylated proteins ........................................ 417 B. ~ of myristoylaSon .............................................. 41S I. Subsu'ate specifkity and intrscellular location .................................. 419 IV. The function 04' protein bound fatty acids ......................................... 419 A. IPalmitoylalioo .......................................................... 419 I. lnvol~-mem in membrane fusion ........................................... 419 2. Moq~zolpmem and protein interaction ....................................... 420 3. Involv~u, mt in pmteolysis protection, transport, cell transformation and silpml t~tion. 420 4. Compm~ of acylat¢d and fatty acid-free peptides ............................. 421 B. Fuucdom of m~rismylation ................................................ 422 1. Anchor func/ica of m~'istoylation .......................................... 422 2. Myzismylation in ~ and virus ~try ................................ 42.3 V. Concludins remazk~ ........................................................ 423 Acknowledgements ............................................................ 423 References ................................................................... 423 !. inlxodbction Many proteins are modified during or after their synthesis. Some of these modifications have been known Abbreviations: VSV, vesicular stomafitis vinms; SFV, Semliki Forest Virus; ER. cndoplasmic r¢ticulum; PAT, protein fatty acyltransferasc: NMT, N-myristoyltrans fm'ase. Correspondence: M.F.G. Schmidt, Kuwait University, Faculty of Medicine. P.O. Box 24923/Code 131.10 S~at, Kuwait, Arabian Gulf. 0167-4157/89/$03.50 ¢ 1989 Plsevier Science Pubfishexs B.V. (Biomedical for many years, as for instance glycosylation, phospho- rylafion and proteolytic cleavage of precursor poly- peptides. These examples have been extensively covered in a number of review articles and in biochemical textbooks over the years. Another type of protein modification discovered more recently is the covalent attachment of lipid molecules like pho: p,h:!ipid, diacylglycerol and various species of long chain fatty acld~. Su,,h bil~ding of lipid molecules is expected to change the physical properties of the re,pective entity quite dramatically, because largely hy- drophilic residues are converted into very hydrophobic Division)
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`412 NH 2 (COOH) (COOH) EXTERNAL fNH~ (,,~H ~, Et..NI~ 0 li~O$itOI LIPID P&LMITOYL&TION MYRISTOY- GLYPI - L&TION &TION Fig. 1. Schematic diagram of hydrophobic modifications of proleins: palmitoylation, tn)'nstoylation and $1ypiation. Cys, cyst¢in©; Oly, glycine; CHO, carbohydrate; Eth-NH:, ethanolamine. Threonine has been identified as the palmhoylation site in one case only, with bovine brain myelin proteolipid apoprotein [90]. All other palmitate linkage sites chen0cally identified are cysteine residues (compare Table n). previously identified that contained fatty acids as part of their covalent structme. These were the proteolipid apoprotein isolated from the myelin membrane, which is also termed lipophilin [5,6], the proteolipid compo- nent of preparations of the sarcoplasmic Ca2+/Mg 2+- ATPase [7] and the bacterial lipoproteins [8,9]. The fatty acid species in these above-mentioned proteins more or less reflected the acyl patterns of the. lipi0s of the membrar.e from which they had been isolated. In all these ca, .:s palmitic-, stearic- and oleic acid were the predomiaant acyl species. Although these proteins, due to their solubility in organic solvents, had initially been termed proteolipids, they can be classified as true acylproteins because of their covalently bound fatty acid residues. It should be mentioned that some other proteolipld species have been described. Despite their lack of any covalently hound fatty acid, these are solu- ble in organic solvents, but insoluble in water [10]. ones. This will, of course influence the interactions between such modified proteins and other molecules present in their vicinity, be it other proteins, lipids or even nucleic acid. Likewise, intermolecular processes may be influenced, such as oligomerisation of poly- peptides during biosynthesis or the co-oIgrativity be- tween subanits of a multimeric protein. The present review aims at summarizing develop- ments in the field of hydrophobic modifications of proteins since their discovery. Special emphasis will be placed on the acylation of viral and eukaryotic poly- peptides with lon~ chain fatty acids. This modification yields what could '~e regarded as a new class of pro- teins, which by analogy with glyco- or phosphoproteins, I suggest by termed 'acylproteins'. Two types of acylprotein are presently distinguished in the literature, those which contain exclusively the fourteen carbon myristic acid (tetradecanoic acid) in an amide-linkage, and those whirl" are predominantly modified with palmitic-, stearic- and oleic acid (hexa- and oc- tadecanoic acids) in ester- or thioester-type linkages. The various biochemical and cell biological aspects of palmitoylatlon and myristoylation will be discussed. Another hydrophobic modification of proteins, the covalent attachment of a glycolipid tail to some mem- brane proteins (glypiatiGn), will not be described here. CmTent reviews cover this area, which the reader is referred to [1-4]. Fig. 1 presents a schematic representa- tion of the above-mentioned hydrophobic modifications of proteins. I!. Ester-type palmJtoylation Even before the term palmitoylation became widely used in the literature, a few protein species had been H-A. Identification of palmitoylated proteins of viral and cellular origin At the time unaware of the above-mentioned acylated proteolipids, Schmidt and Schlesinger [11,12] observed that typical membrane glycoproteins can be labeled with tritiated long chain fatty acids. Although viral membrane $1ycoproteins were utilized initially, the same authors reported that fatW acylation also occurred with proteins of non.infected cells [131. The first cellular membrane glycoprotein to be identified as fatty acyla- tion was the transferrin receptor [14]. Many other species of acylproteins of viral or cellular origin with covalently bound fatty acids were subsequently identified (Table I). In most of the proteins listed in Table I acylatlon was detected after labeling the protein with [~H]pulrnltie acid in the appropriate system followed by identifi- cation via immunoprecipitation, PAGE-analysis, chro- matographic procedures, or combinations thereof. Al- though not examined in all acylproteins listed, in nearly all cases in which labeled fatty acids were analysed, palmitic acid was found to represent the major species bound to the acylprotein with stearic- and olei~ acid comprising the balance of total radioactive fatty acid recovered from the protein under study. The same ,e- suits have also been reported from analysis of acyi chains released from non-labeled acylproteins purified in large scale (for references refer to Table I). It should be noted that proteins modified by a 81ycolipid will incorporate 3H-labeled fatty acids during metabolic labeling, just like the acylproteins. In order to differen- tiate such glypiated proteins from acylproteins, labeling with [3H]ethanolamine or [3H]inositol should be ap- plied. Only if such label is incorporated, is the prott:in most likely 81ypiated [3,4].
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`413 TABLE I Palmitoylated proteins of eukaryotic and viral ortgm Protein species Origin of protein Ref. Cellular acylproteins Transferrin receptor human leukemic T-cells 14 Acetyolcboline receptor" muscle cell line 15 Insufin receptor * human lymphoeytes 16.17 (IM-9). Hep G2 Insulin like growth factor I - receptor Hep G2" MDCK IS IgE receptor leukemic T-cells 19 ~opsin bovine retina 20 Integphotoreceptor bovine retina 21 Na + channel, a-subunit rat neurons 22 Intedeukin-2 receptor human T-cell line (MT-I) 23 Sialo-gp 2,3 and gp3Pr erytht'oblasts 24 HLA B'/. DR lymphoblastoid cell line 25 HLA-D/invariant chain various human lymphoma cells 26 p41 p41 nansfected cell 27 la alpha and beta mouse spleen cells 2g Galactnsyhransferase Hela cells 29 Golgi mannosidase II mouse 31"3 and other cells 30 Cardiac 51 kDa protein mouse 31 Mucus glycoproteins sublingual salivary glands 32 Mucus glycowoteins gastric mucosal cells 33 TGF-alpha precursors TGF.gene transfected cells 34 Butyrophilin milk fat globule membrane 35 (goat) Xanthine oxidase milk fat globule membrane 35 (Soar) [.ipophilin myeSn membrane 0~vine) 5.,5 DM-20 myelin membrane (rat) 36 PO-protein myelin of peripheral nerve (rat) 37 Ca2*-ATIPase sarrmplasmlc reticulum 7,38 Fibmnecdn human flbroblasts 39 Ankyrin human e~jOmxy~es 40 Band 4.1 protein human erytMocytes 40 Vimadin" chicken embryo fibroblasts 41,42 membrane proteins intact oceular lens (rat) 43 Liptin ileal entet~cytes (rat) 44 ras-pgoteins various human cell lines 45 Apolipoproteins (AI.E) human bepatonm cells 46 (Hep-G2) Apolipoprotein B human LDL 47 Folate binding protein* human KB-cells 48 Dcvelopewntal gps sea urchin embryo 49 gAS I and RAS 2 yeast cells 50 YIPTI y e.,~4 cells 51 Alpha factor yea.~t ceils 52,53 Membrane glycoproteins ygast cells M Actin (sub population) slime mold 55~6 Microtubule-binding Trypanesoma brucei 57 protein I..ight hatwestin8 protein" duckweed plant 58 Rib 1,5-Pz-carboxylese" duckweecl plant 58 Six proteins Tettahymena mimbres 59 (major 22 kDa) Suffactant protein alveolar qpith¢lial cells 60 (SAP35) Human tissue factor human lung flbroblast 61 CD9 surface gp human platelets 62 TABLE I (continued) Protein species Origin of protein nef. Viral structural acylproteins G-protein rhabdovirus~s (VSV) I I EI,E2 togavims~ (Sindbis, SFV) t 1,63 HA. HA z avian/human influenza 63 A viruses HA influenTa B virus ~" HEF I. HEF l[ influenza C virus ~' F o. F t Newcastle disease viruses 63 F, HN mumps virus 64 HN SV5 65 E2 mou~ hepatitis vints 66 E2 bovine cotonavims L9 63 GI, G2 bunya viruses (La Crosse) 67 gp 35 Roux sarcoft~ vlru= 68 gp 65 spleen focus fomdng ',rims 69 p37 k vacginia virus 70 gE herpes simplex virus (type I) 71 Viral non-structural aeylproteins Elb 18 kD Adenovirus 12 72 Elb 19 kD Adenovirus i 73 T-antigen (large) SV40 74 ras-protein Harvey murinc sarcoma virus 75 The fatty acid linkage in th¢,~ proteins is parlially or to:ally resistant to treatment with mild alkali of hydgoxylanfine. b Veil. M., Heftier. G. and Schmidt, MF.G.. unpublished data. Many of the palmitoylated proteins listed in Table I are glycosylated and represent membrane components with widely diverse biological functions. Among these are polypeptid~ which span the membrane once or multiple times. Usually the palmitoylated membrane proteins are oriented with their carboxyterminus to- wards th*. cytoplasmic side of the membrane or homolo- gously towards the inside of the enveloped virus par- ticles listed. However, reversely oriented proteins may also be palmitoylated, e.g., the hema881utinin- neuraminidase protein (HN) of mumps virus and of paramyxovirus SV5 [64,65] as well as the transferrin receptor [14]. Some of the membrane proteins listeo occur as monomers [20], others as homo-oligomers [11,631 or as hetero-oligomet~, An example for the latter is the insulin receptor, in which only the ~-subunit is palmitoylated [17,18]. Beside the plasma membrane, internal membranes may also contain proteins with covalently linked fatty acid, e.g., the Golgi-located man. nosidase II and galactosyltransferase [29,30] as well as the proteolipid component of sarcoplasmic Ca 2÷- ATPase [7,38]. Recent more generalized studies of the intracellular location of palmitoylated proteins showed that most of them are membrane bound [76-78]. How- ever, the phenomenon is more complex in a functional sense, since a small group of palmitoylated proteins are secreted by the cells [21,33.34,46,47] and a somewhat larger group of acylproteins comprises components of the cellular cytoskeletal elements [39,40,42,55]. Consid-
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`414 ering palmitoylated proteins of viral origin it can be stated that with the exception of two serotypes of vesicular stomatitis virus (VSV) ['~9] and of Sendal virus [65] (see below) all RNA- and DNA-viruses with a lipid envelope analysed so far contain at least one structural protein with covalently hound palmitic acid. These are all located in the viral envelope, and except for p 37 k of vaccinia virus [70], are also glycosylated, it is note- worthy ~.hat viral spike proteins with a known low pH-dependent fusogenic activity are palmitoylated (see below). With viral non.structural proteins palrnitoylation is not as common a,~ myristoylation (see below). However. cells transformed by the non-enveloped viruses Simian virus 40, adenovirus and with the enveloped Harvey routine sarcoma virus express early proteins which be- come acylated and are located in the plasma membrane of the infected cells [72-75]. where they may play a role in the process of cell transformation (see subsection IV-B). As is apparent from Table I, palmitoylated acylproteins are found in organisms of different levels of complexity, in their evolutionary ra~)ge of occurrence they reach from the fungi Dictyost,.lium and yeast, across the angiosperma (Spirodela o/ sorrhiza ), across the non.vertebrates (sea urchin) up to the vertebrates. mammals and human. As mention¢,i above, viruses from a variety of hosts as well as prokaryotes contain acylproteins. The latter group will not he dealt with, since it has been covered in a recent review by Wu and Tokunaga [80]. Although the list of palmitoylated pro- teins is extensive at this point, more acylated species will probably have to be added once the numerous acylproteins detectable after [)H]palmitoylation in vivo o( various vertebrate cells have been identified 113,8t-831. ll.B. AO'I linkage sites m pa/mitoytated proteins From the early studies of myelin proteolipid [5,6] and of the a~lproteins present in the viral envelope [11,12.63,84]. it is clear that fatty acids are bound by ester-type linkage, since they can be released by mild alkali and hydroxylamine treatment. Stability studies utilizing these agents have si,z~ been applied routinely to identify ester-type acylation of most of the viral or cellular palmitoylated proteins listed in Table I. How- ever, direct structural analysis of the palmitate linkage site proved to he a formidable task. Due to their unusual and unpredictable properties, peptides with covalendy bound fatty acid moieties derived [tom palmitoylated proteins were extremely difficult to purify [12,85]. More indirect studies, as for instance limited proteolysis pointed to a location of the fatty acid linkage site close to the membrane spanning segment of acylated mem- brane proteins [14.63,85-88]. However, despite corn- parative analysis of the stability of the fatty acid linkage i: was initially not even possible, to decide conclusively between an oxygen- and a tbioesfer linkage of thc fatty acid to serine or cysteine, For instance, fatty acids linked to Semliki Forest virus (SFV) El-protein were released with hydroxylamine under the same conditions which cleaved the acetyl-group from o-acetyl serine, suggesting an o-ester linkage between fatty acid and El [89]. However, direct structural analysis has recently revealed a cysteine as the linkage site. and thus a lhioester linkage between protein and fatty acid (see below and gel'. 91). The first direct identification of one of the two palmitoyl linkage sites present in myelin lipoprotein by sequence analysis came from Stoffei and co-workers. who had virtually unlimited supplies of this acylprotein from bovine brain. Their results showed that fatty acid is hound to a threonine residue located in the ex- tracytosolic domain of one of the hydrophilic loops between two of the five membrane segments of lipo- philin [90]. A similar protein chemistry approach was also utilized with three other palmitoylated acylproteins, HLA-D associated invariant (il) chain, VSV G.protein and SFV El-protein. In all three cases palmitic acid was found to he bound to cysteine in thioester linkage. The fatty acylated cysteine of the two viral acylproteins was located on the 'Cytoplasmic' (internal) face of the lipid bilayer with SFV-EI [911 and at the base of the carboxyterminal cytoplasmic tall with the VSV G-pro- tein 1921 (compare schematic cl~ in Fig. 1). Prior to the biochemical identification of fatty acylation sites, other authors had turned to recombinant DNA technol- ogy to locate fatty acids within the primary structure of acylproteins. Replacement of specific cystcine residues suspected to represent the linkage site with serine after site-directed mutagenesis led to a loss of the covalant attachment of fatty acid during in vivo labeling expert. ments with the mutated ras-protein [93,94 I. the ras-like YPTI from yeast 151]. VSV G-protein [95] and the transferrin receptor 1971. The lack of fatty acylation clearly indicated an involvement of the mutated cy- steine residues with fatty acid binding. However, it cot, l~ .~ ~,~ stated with certainty, that those cysteines were the actual linkage sites. Fortunately, the hypoth- esized Cys residues in fas 198] and the G-protein [92] have since been confirmed as fatty acid linkage sites by direct biochemical methods. "/'he results of the various analyses of fatty acid av.achment sites in different palmitoylated proteins are summarized in Table II. Mainly from c~mparison of amino acid sequences around suspected a~Tlation sites of the ras family proteins, a consens~ sequence for palmitoylation has been proposed recently [99]. How- ever, this so called CAAX-box (C for Cys, A for aliphatic and X for any amino acid) at the C-terminus is by
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`TABLE II C).ueme ees,dues as polmatm'lattc~n utes anJ #sear wrox, rw~y " Pahmm~iati~wt ~tc~ arc walked by a.ttc'rtsk~.. 415 Intelp, ad membrane proteins VSV-G NH: ........................................................................................ GLFLVL SFV-E1 NH 1 ................................................................................ VVVTC° IGL Tramfemn-geceptog HOOC ........................................................................... G¥C" 15C, ~,.."" B0~ d~k~in NH2 ........................................................................................... IYIMM HLA-D associated invanant chin li HOOC ............... QYL ................................................................ VLAG P~i~ ~ ixo~cim r~.p¢o~¢in YPTl.pro¢~n (external) (n~'mbrane ~t'lmem I RVGIHLC" IKLK ............................................... COOH RR.COOH KK~KIvNAK .................................................................... NH: NKQFR.~CMVTTLC'C °G .......................................... COOtt RSC*K .............................................................................. NH: HOOC-SLVC * KC ........................................................... NH: HO(;C-C "C * CKTJGTN ...................................................... NH: (~'toplamu¢) • Sequeta:gs are from Refs. 92.95.97.101.26.94 and $! tn tlus orda trc~n top to bottom. definition a rather unprecise recognition sequence. It may be valid for the few pulmitoylated ras-type proteins only. When the primmy structures of other acylated and non-acylated membrane gi)~:oproteins are compared. due to the wide variabifty of amino acids around the putative palmitoylation sites, no recognition sequence can be deduced. The only structural theme common to the pahllitoylated spooks is the occumlcc of at least one cysteine residue usually within about four residues from the putative border between the inter leaflet of the lipid bilayer and th= cytoplasm [6~,|00 i. With the VSV G.protein the pulmitoylated cysteine residue was located seven residu~ into the cytoplasm [921. in a r~.~nt report Ovchitmikov and co-workers identified the acylation sites of bovine duxlopsin. The two fatty acylated cysteir~ residu¢~ were located 13 and 14 amino acid residuet away from the membrane border into the cytoplasm. ~ authors suggest thmt using its fatty acids as anchors, this gegion of the protein loops back into the lipid bilaym of the photofeceptog membrane [1011. il.C. Biochemistry o/palmitoylation il.C I. Location of palmito)'lating acti~,ily From early studies of palmitoylation of viral acylproteins in vivo. it has been apparent that this modification is an event which in the presence of ongo- ing protein synthesis can be detected by metabolic short pulse labeling [102]. in order to define the intracgllular location of palmitoylation, fatty acylation was related to the various stages of oligosaccharide prc~bsing, and to the timing of proteolytic processing and intracellular transport. The results of such experiments revealed that palmitoylation of VSV G-protein and Sindbis El and F,2 occurs shortly after translation and just prior to the acquisition of Endo H resistance [102|. While various trimming inbibitors (swainsonin. deoxynojirimy¢in, castanospermin) either had no effect on palmitoylation (McDowell, W. and Schmidt. M.F.G.. unpublished re- sults) or led to a stimulation [103]. the effect of toni- camycin depended on the 8Jycopwtein under study. While tunicamycin had no effect on the acylation of the HLA invariant-chain [26], coronavirus E2 [104,105] and mannosidase l! [30], it completely abolished palmitoyla- tion of VSV G-protein [12] and of the sodium channel 122l. Thus. it seems that the influence of tunicamycin on acylation is an indirect one. pefltaps by preventing transport of the slycoprotein to the acylation site [106]. This indicates, th~tt glycosylation per sc is not required for fatty acylation. Also other exlx~ments pointed to a crucial intracellular location for paln'itoylation to oper- ate. "rs-mutants of the G.protein defective in transport between the ER and Golgi couM not be acylated at nonpermtssive temperatures !12]. whereas blockin8 of transport bciwggn the Golli and the plasma membrane with rnonensin had no influence on fatty acylation [107]. From pulse.chase experiments with ['lH]palmitic acid Dunphy et al. [1081 and Quinn et al. [109] reposed the cis-Goli~ to be the intraceilular location at which palmitoylatien ofcurs. Subsequently, short pulse label- ing in vivo of acylpmteins with [~H]palmitic acid wzs utilized frequently as a marker for the cis.Gol~ com. partment (e.g., Ref. Ii0). However. mote recent data proved that palm[tic acid binding begins at an even earlier stage. By cell fractionation after extremely sh~
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`416 pulses (20 s) with [3H]palmitic acid in SFV infected bP.by hamster kidney cells (BHK), most of the [3H]palmitoylated F-protein was found in the rough ER fraction [111]. After establishing an in vitro system of acylation, which utilizes deacylated exogenous SFV E1- acceptor protein [54], particular membrane fractions could be tested for protein fatty acyltransferase (PAT). Studies with such a cell-free system using viral acceptors revealed the highest specific activity of PAT in the rough ER fraction [111]. The use of transport mutants revealed that early acylation occurs also with yeast cell giycoproteins [112] and different Ts-mutants of the influenza hemagglutin (Veit, M., SchmidL M.F.(3. and Klenk, H.D., unpublished results). Recently a chimeric construct between a fragment of the influenza hemag- glutinin joined to the C-terminus of nearly compleie rat growth hormone was shown to be palmitoylated. Since this hybrid protein failed to be transported to the C-olgi apparatus, the authors concluded that fatty acid ad- dition occurs in a fraction of the ER which they termed smoot ER cistemae [113]. Although the evidence for early acylaticn of the above proteins seems quite solid, contrasting results were obtained with other cellular acylproteins, as for instance mucus glycoproteins, myelin apolipoproteins and the SV40 large T-antigen. Utilizing an in vitro assay for palmitoylating mucus glycoproteins from gastric mueosa, SIorniany and co-workers reported the Golgi- membrane to be the location of paL'nitoylating ae.fivity [114-116]. However, the membrane fractions in that study were rather crude and had not been tested for marker enzymes. More recent reports on the detection of nascent peptidyl-tRNA with bound fatty acids by the same group, in contrast to their earlier reports, favor a cotranslational acylation of O-glycosylated mucus glycoproteins [117]. Palmitoylation in the vicinity of the late Golgi or the plasma membrane was reported for the large T-antigen [118], the transferrin receptor [119,120,121], myelin lipo- philin [122,123] and bovine rhodopsin [20]. However, with the two latter acylproteins autoacylation was ob- served [124,125,125a], so that in these cases a different mechanism of fatty acid attachment may operate. Some non-enzymatic or autocatalytie acylation has also been observed with F_.2-protein in SFV-infected cells [112]. Such a process may not depend on cellular enzymes, but rather on the availability of acyl-CoA and a receptive conformation of the protein acceptor. The plasma mem- brane as a possible location for palmitoylation of cyto- skeletal proteins resulted from studies with ankyrin in rabbit erythrocytes, which are devoid of nuclei and internal membranes [40,126]. With plant cells, another compartment, the chloroplast, must have palmitoylating activity, since a number of thylakoidal proteins are acylated in that location both in vivo and in vitro (Ref. 58 and Mattoo, personal communication). Only a slight degree of palmitoylation was detected in the fungus Physarium polycep,~alum, but the location of the event was not identified. Interestingly, in this fungus most of the acylated proteins turned out to be myristoylated in amide linkages [127]. Although a definitive general statement on the intra- cellular location of protein acyltransferase is not yet possible, most data available indicate that enzymatic palmitoylation occurs soon after translation. Most likely this modification of proteins operates in an area which could be designated 'late ER', whatever such a location's cell biological features may be. One could speculate that fatty acylation may occur in the first set of vesicles leaving the ER with a cis-Golgi destination. This would fit with the hypothesis that fatty acylation may enhance vesicular transpor! which depends on fusion and fission of lipid membranes (see subsection IV-A1). H-C2. Dynamic nature of palmitoylation Fatty acids bound to acylproteins by ester-type lin- kage are metabolically dynamic. Omary and Trow- bridge [14,119] observed that the transferrin receptor could be labeled with 3H.fatty acid 48 h after its translation, and that the protein bound fatty acids 'turned over' more rapidly than the protein portion of the receptor. Similar observations were aiso reported for bovine rhodopsin [20,126], mannosidase II [30l, ankyrin [12g], ras-protein [129] and a number of cellular acylproteins with unknown iden'.ity [PI]. Recently, dif- ferent half lives were repo~ for the acyl groups of different proteins of red blood cells [129a]. In accor- dance with those observations, Berger and Schmidt [130] detected an enzymatic activity in microsomal membranes of diverse origin, which specifically released fatty acids from exogenously added viral acyiproteins. This protein fatty acylesterase ~quires mild detergents, is completely inhibited at 4 ° or 60°C and by sodium dodecylsulfate, and is clearly distinct from the acylating enzyme protein fatty acyltransferase [130]. The fact that acyl chains can be metabolically released from. palmitoylated proteins may be of some impor- tance, because it allows the regulation of this hydro- phobic modification. Therefore, whatever biological function a given paimitoylated protein may have, it can be switched on and off by fatty acylation or fatty acid release (deacylation by esterase), respectively. In ad- dition to directly modulating biological functions of an acylprotein, specific intracellular locations of an acylprotein could potentially be controlled by acylation and deacylation. Thus, fatty aeylation of proteins could have the same regulatory potential as the well known phosphorylation of polypeptides. 11-C3. Towards the purification of protein fatty acyhrans. ferase (PA 7/') Palmitoylating activity has been measured in various
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`417 cell-free systems utilizing exogenous or endogenous aceeptor proteins [111,112,115,116,123-125,126,128, 131,132]. However, purification of a palmitoylating pro- tein fatty acyltransferase (PAT) has not yet been achieved. Published data on the enrichment of thi~, enzyme are scarce. Partial purification of PAT by cell fractionation and chromatography on hyd.~oxylapatite has been r~.'ported, but no information with regard to structure of the PAT proteins was given [111J30]. In the same reports a PAT assay was used, which required exposure times of several weeks for the det~:t'on of [3H]palmitoylated E1 during SDS-PAGE ~.nalysis and fluorography [111,130]. By improving the cell-free assay for PAT in our laboratory to 500-fold increased sensitivity, fractiona- tion of this activity on various chromatographic col- unms is now possible [132a]. The key features of this new assay are threefold. (1) High specific radioactivity [3H]palmitoyI-CoA prepared by enzymatic synthesis with microbial acyI-CoA synthetase was utilized as lipid substrate. (2) Purified deac~lated and hydroxylamine free El-protein from Sen',:'iki Forest virus was used as exogenous acceptor for fatty acid. (3) Microsomal mem- branes from human placental tissue were identified as a rich source of protein fatty acyltransferase (PAT) (132b). Utilizing this powerful assay, the enzymological charac- terization of PAT as well as its protein chemical charac- terization are presently in progress. Initial results show that PAT is an enzyme of complex structure, which is bound to membranes of the rough ER (Schmidt, M.F.G. and Bums, G.IL, unpublished data). H-C4. Specificity of lipid substrates for ester-type fatty aeylation of proteins Despite this early state of analysis of ester type acylation of proteins, some information is available on its substrate requirements. It has been established that the activated form of fatty acid, acyl-coenzym¢ A, m~cls to be available to allow the acyltransfer onto protein. Alternatively, free fatty acid can also be transferred in the presence of ATP, Mg ~+ and coenmjme A, provided the source of PAT has intrinsic acyI-CoA synthetase activity [115,116,123,133,13,1]. Since a variety of acyl species have been identified in different 'palmitoylated' acylproteins [65,83,135], fatty acyk *ion into es',er-type linkage was not expected to be very ~l~'¢ific with regard to the fatty acyl species. Nevertheless, comparison of acyI-CoA substrates with different chain lengths in cell-free acylation revealed a preference for palmitoyl- CoA [135]. Studies in our laboratory with enriched preparations of PAT revealed that palmitic acid (16:0) is preferred over shorter as well as longer aeyl chains. Likewise, unsaturated acyl species are less well incorpo- rated into E1 acceptor protein when compared to the saturated species of the same chain length (Schmidt, M.P.G., Qanbar, R. and Burns, G.R.. unpublished data). %imilar results were recently repor