M ANY eukaryotic cell-surface proteins are known to

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1 Publshed Onlne: 15 October, 1994 Supp Info: Downloaded from jcb.rupress.org on October 12, 2018 The GPI Anchor of Cell-Surface Protens Is Syntheszed on the Cytoplasmc Face of the Endoplasmc Retculum Jolanta Vdugrene and Anant K. Menon Department of Bochemstry, Unversty of Wsconsn-Madson, Madson, Wsconsn Abstract. Glycosylphosphatdylnostol (GPI) membrane proten anchors are syntheszed from sugar nucleotdes and phospholpds n the ER and transferred to newly syntheszed protens destned for the cell surface. The topology of GPI synthess n the ER was nvestgated usng sealed trypanosome mcrosomes and the membrane-mpermeant probes phosphatdylnostol-speefc phospholpase C, Con A, and protenase K. All the GPI bosynthetc ntermedates examned were found to be located on the external face of the mcrosomal vescles suggestng that the prn- cpal steps of GPI assembly occur n the cytoplasmc leaflet of the ER. Protease protecton experments showed that newly GPI-modfed trypanosome varant surface glycoproten was prmarly orented towards the ER lumen, consstent wth eventual expresson at the cell surface. The unusual topographcal arrangement of the GPI assembly pathway suggests that a bosynthetc ntermedate, possbly the phosphoethanolamne-contanng anchor precursor, must be translocated across the ER membrane blayer n the process of constructng a GPI anchor. M ANY eukaryotc cell-surface protens are known to be anchored n the plasma membrane by covalent lnkage to glycosylphosphatdylnostol (GPI) 1 (for revews see McConvlle and Ferguson, 1993; Englund, 1993). Although the bologcal sgnfcance of proten modfcaton by GPI remans engmatc, emergng clues suggest that GPI-anchored protens may be markers of membrane structural domans that are functonally mportant n ntracellular membrane traffc (Brown, 1992) and transmembrane sgnalng (Brown, 1993). In some polarzed cells and neurons, the GPI anchor also acts as a domnant targetng sgnal servng to delver GPI-anchored protens to specfc plasma membrane domans (Rodrguez-Boulan and Powell, 1992). GPIs have been found n all eukaryotes examned to date, and a wde spectrum of functonally dverse protens rely on a GPI anchor for membrane assocaton. GPIs are constructed n the ER by the acton of at least seven enzymes. The smplest scheme for GPI assembly nvolves sequental addton of components (monosacchardes and phosphoethanolamne) to phosphatdylnostol (PI), leadng to the formaton of a glycolpd wth the mnmal Address all correspondence to Dr. Anant K. Menon, Department of Bochemstry, Unversty of Wsconsn-Madson, 420 Henry Mall, Madson, WI Telephone: (608) ; FAX: (608) Abbrevatons used n thz's paper: BP, heavy chan bndng proten or GRP78; dol, dolehol; EtN, ethanolamne; GleN, glucosamne; GleNAc, N-aeetylglucosamne; GPI, glycosylphosphatdylnostol; Man, mannose; PI, phosphatdylnostol; PI* PI contanng a fatty acd estedfed to nostol; PI-PLC, phosphatdylnostol-specfc phospholpase C; VSG, varant surface glycoproten. structure EtN-P-Man3-GIcN-PI (see Table I). The completed GPI moety s transferred to newly syntheszed protens contanng a carboxyl-termnal GPI-attachment sgnal sequence (Cross, 1990). In parallel wth other modfcatons of translocated protens such as cleavage of the NH2-termnal sgnal peptde (Jackson and Blobel, 1977), N-glycosylaton (Hubbard and Ivatt, 1981), and formaton of dsulfde bonds (Lambert and Freedman, 1983), t s wdely assumed that GPI attachment to the polypeptde chan occurs n the ER lumen (Amthauer et al., 1993). The recpent protens are targeted durng synthess to the ER va a conventonal NH2-termnal sgnal sequence and translocated across the ER membrane blayer. GPI s then attached (va ethanolamne) to the carboxyltermnal amno acd exposed upon removal of the carboxyltermnal sgnal peptde. By analogy wth other ER glycosylaton reactons, most notably those nvolved n the fnal stages of assembly of the dolchol (dol)-lnked olgosaccharde precursor of N-lnked sugars, t has also been suggested that the assembly of the GPI structure occurs n the lumenal leaflet of the ER membrane blayer gvng rse to a product approprately postoned for transfer to proten (Menon et a., 1990b; Abejon and Hrschberg, 1992). Despte consderable progress n elucdatng the bochemcal steps nvolved n the constructon of the GPI moety, evdence concernng the topology of ths pathway s lackng. The phospholpd substrate, PI, and the drect reacton donors (UDP-GlcNAc, dol-p-man, and phosphatdylethanolamne [Doerng et al., 1989; Menon et al., 1990b; Menon et al., 1993]) requred for GPI assembly are syntheszed n the cytosol or n the cytoplasmc leaflet of the ER membrane The Rockefeller Unversty Press, /94/10/333/9 $2.00 The Journal of Cell Bology, Volume 127, Number 2, October

2 blayer, but can be transported to the ER lumen (Bell et al., 1981; Hutson and Hggns, 1982; Haselbeck and Tanner, 1982; Hggns et al., 1989; Perez and Hrschberg, 1985; Abejon and Hrschberg, 1992). Thus, based on substrate avalablty, GPI assembly s not restrcted to ether sde of the ER membrane. Recent data show that early GPI bosynthetc lpd ntermedates (GlcNAc-PI and GlcN-PI, see Table I) are located n the external membrane leaflet of ER vescles suggestng that GPI assembly s ntated n the cytoplasmc leaflet of the ER (Vdugrene and Menon, 1993). Sequences of genes encodng protens nvolved n GlcNAc transfer to PI are less nformatve but are nevertheless consstent wth ths hypothess (Knoshta and Takeda, 1994). No nformaton s avalable on the topology of the reactons nvolved n elaboratng GIcN-PI to generate the GPI anchor structure. In ths paper, we show that the prncpal steps of GPI assembly are confned to the cytoplasmc face of the ER membrane. It seems lkely that the product of these reactons, a phosphoethanolamne-contanng GPI, s then translocated across the blayer nto the lumenal leaflet for transfer to proten. Materals and Methods Materals GDP-[2-3H]mannose and UDP-[6-3H]GIcNAc (20 C/mmol) were purchased from DuPont-New England Nuclear (Boston, MA). Protenase K and Staphylococcus aureus nuclease were obtaned from Boehrnger Mannhelm Corp. (Indanapols, IN). UDP-GIcNAc, GDP-mannose, ATE methylt~-o-mannopyranosde, Con A (type VI) were from Sgma Chem. Co. (St. Lous, MO). Tuncamycn was purchased from Calbochem-Bchrng Corp. (La Jolla, CA). Bacllus thurngenss phosphatdylnostol-specfc phospholpase C (PI-PLC) was a gft from Dr. Martn G. Low (Columba Unversty, New York), and antbodes rased aganst Trypanosoma bruce heavy chan bndng proten or GRP78 (BP) were generously provded by Dr. James D. Bangs (Unversty of Wscensn-Madson, Madson, WI). The Rad-FreC" kt for chemlumnescent detecton of Western blots was obtaned from Schlecher & Schuell (Keene, NH), and slca 60 thn layer plates were from Merck (Gbbstown, N J). Preparaton of Trypanosoma bruce Mcrosomes Bloodstream forms of T. bruce stran 427 (clone 117) were solated from the blood of nfected rats as descrbed p~vously (Feld and Menon, 1992). Purfed trypanosomes were resuspended n BSA-contanng RPMI-1640 medum at gl0 a cells/nl, and ncubated wth tuncamycn (400 ng/ml) for ran at 37 C. The cells were washed twce wth PBS, and membrane fractons were prepared after dsruptng the cells by hypotonc lyss (Bangs et al., 1993) or ntrogen cavtaton (Vdngrene and Menon, 1993) after prevously descrbed procedures. Brefly, washed cells were resuspended at x 109 ceus/ml n hypotonc buffer (1 mm Hepes/NaOH ph 7.5, 1 mm EDTA), and, after a 10-mn ncubaton on ce, the lysate was passed through a 25 gauge needle three tmes. The lysate was adjusted to sotoncty by addng 10 concentrated buffer A (buffer A: 25 mm Hepes/NaOH ph 7.5, 0.25 M sucrose, 1 #g/ml leupeptn, 0.1 mm TLCK, 1 mm DTT) and cell dsrupton was completed wth fve strokes n a Dounce homogenzer equpped wth a tght fttng pestle. Alternatvely, washed cells were resuspended n buffer A at 91-2 x 109 cells/ml, and dsrupted by two cycles of ntrogen cavtaton (400 ps N2 pressure), n a mn-bomb dsrupton chamber (Kontes, Vneland, NJ) followed by three strokes of a tght pestle Dounce homogenzer. After lyss, cell ghosts, nucle, and mcrobodes were removed by low speed centrfugaton (1,000 g for 10 ran at 4oc) and the resultng "postnuclear" supernatant was fractouated by centrfugaton at hgh speed (Beckman 70T rotor, 43,000 rpm, 2 h, 4 C) usng ether a dscontnuous sucrose gradent (steps at 38%, 30%, and 20% sucrose, wt/vol) or a smple sucrose cushon (0.5 M) prepared n buffer A. In each case the pellet was resuspended n barfer A (4-5 X 109 cell equvalents [cell eq]/ml), frozen mmedately, and stored at -70 C. The two preparatons were used nterchangeably n the experments reported n ths paper. In Vtro Bosynthess of GPIs GlcNAc-PI and GlcN-PI were syntheszed by ncubatng trypanosome mcrosomes (typcally 2.5 x 107 cell exl) wth UDP-[3H]GIcNAc ( ~C) n 50 #1 buffer A contanng 5 mm EDTA at 37*(2. At the end of the ncubaton, samples were placed on ce and lpds were extracted as descrbed (Stevens and Raetz, 1991). Lpd extracts were analyzed by TLC on slca 60 usng ehloroform-mohanol-1 M NH_4OH (10:10:3, by volume). After chromatography, the plates were ar dred and scanned for radoactvty wth a Berthold LB 2842 automatc scanner (Berthold Analytcal Instruments, Inc., Nashua, NH). Incorporaton of radoactvty nto ndvdual lpd speces was determned usng ntegraton software suppled wth the scanner n conjuncton wth lqud scntllaton countng. For pulse-chase experments wth UDP-[3H]GIcNAc, the membranes (0.5 x 10 s cell cq) were labeled wth 1 #C of UDP-[3H]GIcNAc n 75 #1 buffer B (buffer A + 5 mm MgC12, 5 mm MnCI2, I mm ATP, I mm CoA, 200 ng/ml tuncarnycn) for 10 ran at 37 C. Then, non-radoactve sugar nucleotdes (GDP-mannose and UDP-GlcNAc, 1 mm each fnal concentraton) were added and the samples were ncubated further for 0-45 mn as ndcated. At the end of the ncubaton lpds were extracted and analyzed as above. Mannosylated GPI precursors were syntheszed and analyzed as descrbed prevously (Feld and Menon, 1992). Brefly, trypanosome mcrosomes (2-5 x 107 cell eq) were ncubated wth #C of GDP- [3H]mannose and 1 mm non-radoactve UDP-GlcNAc n /~1 buffer 2 for 5-60 ran at 37 C. Where ndcated, 0.5 mm PMSF was ncluded n the labelng medum. The reacton was stopped by placng the sample on ce and lpds were extracted n chloroform-methanol-water (fnal composton 10:10:3, by volume). The extract was desalted by butanol-water parttonng and analyzed by TLC (slca 60, chloroform-methanol-water, 10:10:2.5, by volume). PI-PLC Treatment of Mcrosomes: Analyss of GPI Hydrolyss PI-PLC (1,700 U/ml, 1 U = the amount of enzyme capable of hydrolyzng 1 #mol of pbosphatdylnostol per mnute at 37 C n ph 7.0 buffer contanng 0.1% sodum dcoxycholate [Low, 1992]) was obtaned from Dr. M. G. Low (Columba Unversty, New York, NY). Mcrosomes radolabaled va GDP-[3H]mannose or UDP-[3H]GIcNAc were placed on ce; PI-PLC (0-5 U/ml) was added and after ncubaton on ce (typcally 20 ran), lpds were extracted and analyzed as descrbed above. No hydrolyss was observed durng lpd extracton,.e., after addton of organc solvents to the sample. Con A Bndng to Mannosylated GPIs Extracts contanng [3H]Man- or [3H]GlcN-labaled GPIs were dred and the resdue was dssolved n buffer C (25 ram Hepes/NaOH ph 7.5, 0.1 M sucrose, 150 mm KCI, 5 mm MgC12, 5 mm MnCI2) contanng 0.5% Trton X-100). Alquots of the lpd soluton (20 #1, 5, cpm) were mxed wth dfferent dlutons of Con A (80 #1, 0-6 rng/ml prepared n buffer D: 25 mm Hepes/NaOH ph 7.5, 150 mm KCI, 5 mm CaC12). After 20 ran on ce, the samples were processed by addng 5 #1 of 0.5 M methyl ot-dmannopyranosde (to block further bndng), 50 #1 of calf serum (to ensure 100% precptaton of organc nsoluble materal durng lpd extracton [see below]) and 95 #1 buffer D. Lpds were extracted by addng 1.7 ml chloroform-methanol (1:1, by volume) to generate a sngle organc phase (Masterson et al., 1989; Feld and Menon, 1992). Insoluble materal (serum protens, Con A, and Con A-lpd complexes) was removed by centrfugaton (1,500 g, 20 mn, 4 C) and the superuatant was carefully removed, dred, and analyzed by TLC as descrbed above. GPIs recognzed by Con A are expected to precptate wth the lectn durng lpd extracton and hence be depleted from the lpd extract. For experments n whch Con A was tested for ts ablty to bnd GPIs n ntact mcrosomes, radolabeled GPIs were syntheszed by ncubatng trypanosome membranes wth GDP-[3H]mannose n buffer C as descrbed above. Alquots of the labeled membranes (typcally 2 x 107 cell eq n 20 ~1) were mxed wth dlutons of Con A (80 ~1, prepared n buffer D) and buffer D, and after a 20-ran ncubaton on ce, lpds were extracted nto chloroform-methanol-water (10:10:3, by volume) and analyzed as descrbed above. Integrty of ER Vescles The ntegrty of solated trypanosome mcrosomal vescles was determned by protease protecton experments usng BE an ER lumenal proten, as The Journal of Cell Bology, Volume 127,

3 the reporter. Membranes (2.5 x 10 cell eq n 100/~1 buffer B) were mocklabeled for 30 ran at 37"C and placed on ce. Alquots of the sample (10 t*l) were then dluted wth 90 $1 25 mm Hepes/NaOH ph 7.5, 0.25 mm sucrose wth or wthout protenase K and Trton X-100 (50 tlg/ml and 0.5 % fnal concentratons, respectvely). Where ndcated, the membranes were treated wth PI-PLC (on ce or at 37 C) before protenase K addton. After 30 mn on ce, proteolyss was stopped by addng 300 t~l of 4 mm PMSF n water (dluted from a 0.2 M stock soluton n 95% ethanol just before use). Protens were precptated by addng 400 #1 of ce-cold 14% (wt/vol) TCA. After a 30-mn ncubaton on ce, the sample was centrfuged at 12,000 g for 15 mn at 4 C. The pellet was washed wth 500/~ ce-cold acetone contanng 1% TCA, left at -70 C for 30 mn, and then centrfuged agan. The pellet was resuspended n SDS-PAGE sample buffer and analyzed by 10% SDS-PAGE. After electrophoretc separaton, protens were transferred to Rad-Free TM membrane usng standard procedures. The blots were probed wth antbodes rased aganst Torpanosoma bruce BP (Bangs et al., 1993) and alkalne phosphatase-conjugated secondary antbodes. Immunoreactve bands were vsualzed by addng a chemlumnescent alkalne phosphatase substrate suppled wth the Rad-Free TM kt and exposng the blots to X-ray flm. At least two dfferent flm exposures were obtaned to verfy lnearty of the flm response for denstometrc analyss. Denstometry was performed by Kendrck Laboratores (Madson, WI) usng a LGS-50 Laser Scannng Denstometer and QGEL software. Transfer of GPls to Endogeneous Proten Accepters Trypanosome mcrosomes (10 s cell eq) were ncubated at 37 C wth 1.5 /~C GDP-[3H]mannose and 1 mm non-radoactve UDP-GIcNAc n 100/~1 buffer B. For protease protecton experments, labeled mcrosomes were subjected to protenase K treatment as descrbed above, before analyss. 10% of the sample was taken for lpd extracton and TLC. The remander of the sample was analyzed by SDS-PAGE and fluorography (usng EN3HANCE, DuPont-New England Nuclear). Exposure tmes of "~ 2 wk were usually requred, and at least two dfferent flm exposures were obtaned for denstometrc analyss. Denstometry was performed as descrbed above. Results In Vtro Synthess of Radolabeled GPIs Labelng strateges were developed to probe the transblayer dstrbuton of GPIs n the ER membrane blayer. Radolabeled GPIs were syntheszed by ncubatng trypanosome mcrosomes wth radoactve sugar nucleotdes (UDP-pH]- GlcNAc or GDP-pH]Mannose) n the presence of tuncamycn (to prevent synthess of dolchol-pp-olgosacchardes derved from dolchol-pp-glcnac). Incubatons were performed at 37 C. No ncorporaton of radoactvty was seen when mcrosomes were ncubated wth radolabeled precursors on ce, nor dd the spectrum or ntensty of the radolabeled GPIs change when a labeled sample was mantaned on ce (not shown). In some experments GPI bosynthess was blocked at the GlcN-PI stage or the Man3GlcN-PI stage by ncludng EDTA or PMSF n the labelng medum (Vdugrene and Menon, 1993; Masterson and Ferguson, 1991). At the end of the ncubaton lpds were extracted and analyzed by thn layer chromatography. The varous labeled GPIs were dentfed by TLC moblty and by dagnostc treatments, ncludng hydrolyss by PI-PLC. Representatve chromatograms from GDP-[3H]mannose - or UDP-[3H]GIcNAc - labelng experments are shown n Fg. 1. The structures and propertes of the radolabeled GPIs are summarzed n Table I. Non-Mannosylated GPI Lpds Are Located n the External Leaflet of the Mcrosomal Membrane Blayer Fg. 2 A shows that sgnfcant amounts of radolabeled O..= "rj A P2 M2 B C M2 1 + M3* 2* 1 + M3* t I J GlcN-PI l dstance [can] GlcNAc-PI g f3 t2 '--o "0 Fgure 1. Thn layer chromatograms of n vtro labeled GPIs. Radolabeled GPIs were syntheszed by ncubatng trypanosome mcrosomes wth GDP-[3H]-marmose (A and B) or UDP- [3H]GIcNAc (C). Assay mxtures contaned ether no nhbtors (A), or PMSF to block the GPI ethanolamnephosphotransferase (B), or EDTA to block mannosylaton (C). Lpds were extracted and analyzed by TLC usng chloroform-methanol-water, 10:10:2.5, by volume (A and B) or chloroform-methanol-1 M NIL,OH (10:10:3, by volume) (C). The dstrbuton of radoactvty n the chromatograms was vsualzed usng a TLC scanner. Labelng condtons were as follows. (.4 and B) 0.2 /zc GDP-[3H]marmose, 2 x 107 cell eq of mcrosomes n 20 /zl buffer B (+0.5 mm PMSF), 20-mn ncubaton, 37 C; (C) 0.35 /zc UDP-pH]- GIcNAc, 3 x 107 cell eq of mcrosomes n 50 #1 buffer A (+0.5 mm EDTA), 30-mn ncubaton, 37 C. Arrowheads denote the orgn and solvent front. Lpd nomenclature s gven n Table I. mcrosomal GIcNAc-PI and GIcN-PI are hydrolyzed on exposng trypanosome mcrosomal vescles to relatvely low concentratons of PI-PLC on ce (~75 % hydrolyss after 30 mn wth 1.7 U/ml PI-PLC). Snce the mcrosome preparaton conssts of a populaton of sealed vescles (see below) and PI-PLC cannot traverse the membrane, these data ndcate that GlcNAc-PI and GlcN-PI are prmarly located n the external (cytoplasmc) leaflet of the mcrosomal vescle membrane. We next nvestgated whether these cytoplasmcauy orented pools of GIcNAc-PI and GlcN-PI represent bosynthetcally actve precursors that are used n subsequent steps of GPI assembly. EDTA was omtted from the reacton mxture, and converson of GlcNAc/GlcN-PI nto mannosylated GPI speces was measured usng a pulse-chase protocol. The membranes were labeled wth UDP-pH]GlcNAc for 10 ran, and then chased by addng a 1,000-fold excess of nonradoactve UDP-GIcNAc and GDP-mannose. As expected 20 ~2 '-'0 Vdugrene et al. Glycosylphosphatdylnostol Anchor Assembly 335

4 Table I. In Vtro Radolabeled GPIs PI-PLC Con A Lpd Structure susceptblty bndng GIcNAc-PI GIcNAc-PI + - GlcN-PI GIcN-PI + - M 1 ManlGlcN-PI + - M2 Man2GIcN-PI + + M3 Man3GIcN-PI + + M2* Man2GIcN-PI* - ND M3* Man3GIcN-PI* - + P2 EtN-P-Man3GlcN-PI + + P3 EtN-P-Man3GIcN-PI* - + The dfferent lpds are bosynthetc ntermedates representng partal structures and/or modfed versons of the core GPI moety: EtN-P-6Manot 1-2Manotl- 6Manal-4GlcNt~l-6lnostol-P-lpd (Masterson et al., 1989; Menon et al., 1990a). GPIs contanng PI* are resstant to hydrolyss by PI-PLC (Roberts et al., 1988; Krakow et al., 1989; Mayor et al., 1990). ND, not determned. (Masterson et al., 1989; Menon et al., 1990a), radoactvty n GlcNAc-PI and GlcN-PI was rapdly lost durng the chase (Fg. 2 B, closed squares) as the precursors were converted nto mannosylated GPI speces (Fg. 2 B, open squares). Interestngly, topologcal analyss of GlcNAc/GlcN-PI durng the chase ndcated that loss of radoactvty occurred manly n the PI-PLC-senstve pool (Fg. 2 C, open damonds); changes n the amount of the PI-PLC-resstant fracton (Fg. 2 C, closed damonds) were mnor. These data are consstent wth the hypothess that cytoplasmcally orented GlcN-PI s a substrate for the frst GPI mannosyltransferase and that mannose transfer probably occurs on the external (cytoplas- 40 N- '~ 3o 20 z lo o A 1'0 1'5 2~ 2'5 30 tme (mnutes) 60/ I 60 ~" ~4o t ~ ~o~ z 2o... I o ; cl 01,,. I chase tme (mnutes) Fgure 2. PI-PLC hydrolyss of GIcNAc-PI and GIcN-PI n ntact mcrosomes. (A) Mcrosomes were labeled wth UDP-PH]GIc- NAc as descrbed n Fg. 1 C, and then placed on ce and treated wth 0.34 U/ml or 1.7 U/ml PI-PLC for dfferent tmes. Lpds were extracted and taken for lqud scntllaton countng and TLC analyss. (B and C) Merosomes were ncubated wth 1 #C UDPpH]GIcNAc for 10 ran at 37 C n 75 #1 buffer B, and then chased wth non-radoactve UDP-GIcNAc. Lpds were extracted drectly or after treatment wth PI-PLC (0.34 U/ml, 20 ran on ce). B ndcates the total amount of radoactvty n GIcNAc-PI and GIcN-PI (sold squares) and n rnannosylated GPIs (M-GPI, open squares). C ndcates the result of PI-PLC analyss of mcrosornal GleNAc- PIfGlcN-PI at each pont n the chase: radoactvty n the PI-PLC accessble pool of GlcNAc/GIcN-PI s ndcated by the open damonds and resdual radoactvty n GIcNAc/GIcN-PI alter PI-PLC treatment s ndcated by the closed damonds. mc) face of mcrosomal vescles. More elaborate explanatons of ths result nclude the possblty that cytoplasmcally orented GlcN-PI flps nto the lumenal leaflet durng the chase, and s converted to M1 at about the same rate to gve a constant steady-state amount of PI-PLC resstant GlcN-PI. The PI-PLC-resstant fracton of GlcNAc/GlcN-PI represents a pool of lpd that s protected from enzyme acton. The ER membrane s an obvous permeablty barrer and the PI-PLC-resstant fracton could correspond to a lumenal pool of GIcNAc/GIcN-PI. On the other hand the PI-PLCresstant fracton may represent cytoplasmcally orented GIcNAc/GlcN-PI molecules that are located n stercally constraned envronments whch prevent PI-PLC access; sterc constrants could be generated through non-specfc nteractons wth other lpds or protens, or specfc nteractons wth protens such as the varous transferases nvolved n constructng the GPI glycan. Ths nterpretaton s supported by speculaton elsewhere that sterc constrants are probably responsble for the neffcent cleavage of membrane-bound GPI-anchored protens by PI-PLC (Low and Kncade, 1985; Nagel and Boothroyd, 1989), although n these cases the GPI-lnked polypeptde chan tself may be a factor n hnderng enzyme access. These hypotheses concernng the PI-PLC-resstant fracton of mcrosomal GIc- NAc/GlcN-PI are not mutually exclusve and nether can be drectly ruled out n our experments. Sgnfcant Amounts of PI-PLC-Senstve Mannosylated GPIs (M2, M3, and P2) Are Located n the External Leaflet of Mcrosomal Vescle Membranes To characterze the topologcal dstrbuton of the GPI mannosylaton reactons further, we used PI-PLC to probe the dstrbuton of mannosylated GPI speces n mcrosomal vescles. M2, M3, and P2 were examned as these lpds are substrates for PI-PLC n detergent solutons. We dd not study the dstrbuton of a fourth PI-PLC-senstve lpd, M1, as ths lpd chromatographs dentcally wth PI-PLCresstant M3* n several TLC systems that we tested (Menon et al., 1990a). The M3 GPI speces was probed n PMSFtreated mcrosomes (see Fg. 1 B); the other mannosylated GPI speces were labeled at suffcently hgh levels for analyss n normal mcrosomes (Fg. 1 A). Mcrosomes were ncubated wth GDP-pH]mannose n the presence or absence of PMSF for 20 ran at 37 C, and then treated wth dfferent concentratons of PI-PLC for 20 mn on ce before lpd extracton and analyss. Fg. 3 A shows that up to 65 % of M3 and 60% of M2 n PMSF-treated mcrosomes were hydrolyzed by PI-PLC (1-1.5 U/ml). Smlar experments performed n the absence of PMSF showed approxmately the same level of hydrolyss of mcrosomal M2 ( % (n = 11)) by 1.7 U/ml PI-PLC (Fg. 3 B). The transblayer dstrbuton of mcrosomal P2 was also determned n the same experments. Fg. 3 B shows that,o50% of n vtro syntheszed P2 was hydrolyzed by PI-PLC (1.7 U/ml) under standard condtons (see also Table II). The average percent hydrolyss of mcrosomal P2 determned from several experments was % (n = 11). Snce the three lpds are hydrolyzed effcently (80-90%) once the mcrosomes are solublzed n detergent, the ncom- The Journal of Cell Bology, Volume 127,

5 9O 75 6O 45 3O A ~M: ~M: B ~ M2 p2 Table II. PI-PLC and Con A Interact wth the Same Pool of Mcrosomal GPIs % lpd depleted PI-PLC (U/ml) Con A (mg/ml) P2 M PI-PLC (unts/ml) Fgure 3. PI-PLC hydrolyss of n vtro labeled mannosylated GPIs n ntact mcrosomes. Mcrosomes were ncubated wth GDP- [3I-I]mannose n the presence (A) or absence (B) of PMSF as descrbed n Fg. 1 A and B, and then treated wth dfferent concentratons of PI-PLC for 20 mn on ce. Lpds were extracted and analyzed as descrbed n Materals and Methods. 10% of the lpd extract was taken for lqud scntllaton countng. The remander of the sample was dred, dssolved n 20 #1 water-saturated butanol, and analyzed by TLC as n Fg. 1 A. Lpd hydrolyss was calculated from the amount of radoactvty ncorporated nto ndvdual lpd speces before and after PI-PLC treatment. plete hydrolyss observed n our experments suggests that there may be lumenal pools of the lpds protected from enzyme acton by a membrane barrer, and/or that PI-PLC hydrolyss of lpds located n the external leaflet of the mcrosoma membrane s neffcent under the expermental condtons employed. As dscussed above, nether explanaton can be ruled out. It was possble to ncrease the level of hydrolyss (up to 90% and 75% for M2 and M3, respectvely) whle retanng mcrosomal vescle ntactness (see below) by performng PI-PLC ncubatons at 37 C, but uncertantes n assessng the contrbuton of ongong lpd metabolsm and the effect of PI-PLC on the flux through the bosynthetc pathway under these condtons make the nterpretaton of ths result unclear. Nevertheless, the overall data ndcate that sgnfcant amounts of all three mannosylated GPIs (M2, M3, and P2) are located n the external (cytoplasmc) leaflet of the mcrosomal membrane blayer. Intactness of Mcrosomal Vescles Verfcaton of the ntactness of the mcrosoma membrane barrer s crtcal for the nterpretaton of the experments descrbed above. Snce GPI bosynthess occurs n the ER (Vdugrene and Menon, 1993), t was mportant to determne the qualty of the ER vescles n the mcrosomal fracton. The ntactness of the ER-derved mcrosomal vescles before and after PI-PLC treatment was montored by determnng the extent to whch a lumenal ER proten, BP, was protected from the acton of exogenously added protenase K. Publshed data show that very lttle BP s released durng hypotonc lyss and mcrosome preparaton (Bangs et al., 1993) ndcatng that dsrupton of the mcrosomes durng these procedures s mnmal. Fg. 4 shows that BP (determned by SDS-PAGE, Western blottng and denstometry) was quanttatvely protected from proteolyss n the absence of detergent. Importantly, mock-ncubaton of the mcro- Mcrosomes (2.5 x 107 cell eq) were ncubated wth 0.25/~C GDP-[3H]man - nose n 20 t~l buffer C for 20 ran at 37"C. PMSF (0.5 ram) was ncluded to obtan data on M3. After labelng, the membranes were treated wth PI-PLC on ce n the presence of dfferent concentratons of Con A (20-mn ncubaton) and lpds were extracted as descrbed n the Con A-bndng protocol (Materals and Methods). The results are presented as the % lpd (P2 or M3) lost from the lpd extract due to PI-PLC hydrolyss and/or Con A bndng. somes at 37 C followed by PI-PLC treatment on ce had no measurable effect on BP proteolyss. Smlar protease protecton results were obtaned f the mcrosomes were treated wth PI-PLC for 30 mn at 37 C (not shown). These data ndcate that the mcrosomal vescles are ntact and that ther qualty s unaffected by PI-PLC treatment, valdatng the use of PI-PLC as a probe of transblayer lpd dstrbuton. Both PI-PLC-Senstve and Resstant Mannosylated GPIs Bnd Con A n Intact Mcrosomes The transmembrane dstrbuton of labeled GPIs was nvestgated wth Con A as a membrane-mpermeant probe, a technque that has been successfully used to determne the orentaton of dolchol-lnked olgosacchardes n mcrosomal vescles (Snder and Robbns, 1982; Snder and Rogers, Fgure 4. Proteolyss of BP n ntact and detergent-solublzed mcrosomes. Mcrosomes were treated wth protenase K (50 #g/ml) n the absence or presence of a membrane-dsruptng concentraton of Trton X-100. Where ndcated, the mcrosomes were ncubated at 37 C for 30 mn followed by PI-PLC treatment (1.5 U/ml) for 20 mn on ce (to mmc GPI radolabelng and PI-PLC treatment procedures), before protenase K was added. BP was probed by Western blottng. Denstometrc analyss gave the followng results (n arbtrary unts): (Lane 1, control) 40420; (lane 4, +mock ncubaton, +protenase K) (120% of the control). Vdugrene et al. C-tycosylphosphatdylnostol Anchor Assembly 337

6 -~ 6O -9, 4O < u 2O A l Con A (rng/ml) P2 ' P3 ~M3 M2 tlmeman I- before j ConA 60 ~.40 < 20 ~ B )M Con A (mg/ml) Fgure 5. Con A bndng to GPIs. (A) [3H]Mannose-labeled GPIs (10,000 cpm/20 #1 buffer C) were ncubated wth dfferent amounts of Con A for 20 mn on ce. The reacton was stopped wth 5 #1 0.5 M methyl a-d-marmopyranosde (ameman) and GPIs were extracted as descrbed n Materals and Methods and analyzed by lqud scntllaton countng and TLC. In control assays, ameman was added to the sample before Con A addton. (B) Mcrosomes (2 x 107 cell eq n 20 #1 buffer C) were ncubated wth 0.2 #C GDP- [3H]mannose for 20 mn at 37 C, and then treated wth dfferent concentratons of Con A for 20 ran on ce. Lpds were extracted and analyzed as descrbed n Materals and Methods. The extent of the Con A-bndng reacton was determned from the amount of ndvdual lpds recovered n the organc extract. 1984). Snce prevous work (Bangs et al., 1988) showed that proten-lnked GPIs bnd to Con A, we frst needed to determne whch of the GPI bosynthetc ntermedates would also bnd. Detergent solutons of radolabeled GPIs were ncubated wth dfferent amounts of Con A on ce, and then lpds were extracted and analyzed. Durng lpd extracton, GPIs recognzed by Con A are expected to precptate wth the lectn, whereas GPI speces not bound to Con A are expected to be recovered quanttatvely n the organc extract. As shown n Fg. 5 A, all GPI speces wth three marmose resdues (M3, P2, and P3) were sgnfcantly depleted (60-70 % maxmum effcency) from the lpd extract ndcatng that they were bound to the lectn. The bndng effcency was unaffected by nostol acylaton as P2 and P3 were equally well recognzed. Bndng of the M2 speces was much weaker, and bndng of all speces was specfcally nhbted by ~D-methylmannopyranosde (ct-meman). These results are consstent wth the known structural requrements for Con A-lgand bndng (Ogata et al., 1975) and ndcate that GPI bosynthetc ntermedates are specfcally recognzed by Con A. To determne the extent to whch mcrosomal GPIs bnd Con A, GDP-[3H]mannose-labelcd membranes were ncubated on ce wth dfferent amounts of Con A. Lpds were then extracted and analyzed by TLC. Fg. 5 B shows that sgnfcant amounts (55, 65, and 30%) of P2, M3, and P3 were depleted from lpd extracts of Con A-treated mcrosomes ndcatng that all three lpds are accessble on the cytoplasmc face of the rncrosomal vescles. The results for P2 and M3 are quanttatvely smlar to those obtaned wth PI- PLC. Ths observaton s substantated by the results summarzed n Table 11. When Con A (up to 5 mg/ml) was ncluded n standard PI-PLC treatments of labeled mcrosomes (1.7 U/ml PI-PLC, 20-ran ncubaton on ce), very lttle (<10%) addtonal P2 or M3 was depleted from the lpd extract nd- )P2 P3 )M2 carng that both probes nteract wth the same mcrosomal pool of P2 and M3. An addtonal pont of nterest concerns the sgnfcant dfference n Con A reactvty of mcrosomal P2 and P3 (Fg. 5 B). Snce both P3 and P2 bnd Con A equally well n detergent soluton (Fg. 5 A), the dfference n bndng seen wth ntact vescles suggests that sgnfcantly larger quanttes of P3-compared to P2-may be localzed n the lumenal leaflet of the ER. The Con A/PI-PLC experments also generated nformaton on M1 and M3* (Table I), two GPI structures that we were unable to study convenently usng PI-PLC alone because they comgrated n several TLC systems. M1 s PI- PLC-senstve but s not recognzed by Con A; M3* s PI- PLC-resstant but Con A reactve. In PMSF-treated mcrosomes, 56% of M1/M3* was hydrolyzed by PI-PLC (1.7 U/ml), 30% was bound to Con A (5 mg/ml), and 76% was depleted by a combnaton of the probes. Although the precse proporton of M1 and M3* n M1/M3* s unknown, these data ndcate that at last 80% of M1 and 68% of M3* s located on the external face of the mcrosomes. Data obtaned from mcrosomes wthout PMSF were smlar, ndcatng dstrbutons of >63 % for M1 and >65 % for M3* n the external leaflet of the vescle membrane. Newly GPI-modfed Trypanosome Varant Surface Glycoproten Is Located n the Lumenal Leaflet of ER Mcrosomes Mature phosphoethanolamne-contanng GPIs are transferred to newly syntheszex protens possessng a carboxyltermnal GPI-attachment sgnal sequence. It has been shown prevously that trypanosome varant surface glycoproten (VSG) polypeptdes left unprocessed at the tme of cell lyss and mcrosome preparaton are capable of beng modfed by n vtro syntheszed GPIs (Mayor et al., 1991). We have taken advantage of ths endogenous pool of acceptors to nvestgate the topology of GPI transfer to proten. Mcrosomal membranes were radolabeled wth GDP-[3H]man - nose and TCA-nsoluble materal was analyzed by SDS- PAGE. The transfer of a GPI anchor to VSG was followed by assessng the ncorporaton of [3H]mannose nto the polypeptde chan. Snce these experments were carred out wth mcrosomes prepared from tunlcarnycn-treated cells and by ncludng tuncamycn n the assay mxture, no proten-assocated radoactvty can be ascrbed to N-glycosylaton (Mayor et al., 1991). As shown n Fg. 6, only one major radolabeled band of 55 kd was detected, correspondng to trypanosome VSG (varant 117). To examne the localzaton of n vtro labeled VSG, we tested the extent to whch the labeled VSG polypeptde was protected from exogenously added protenase K. Fg. 6 shows that the majorty (73 %) of newly GPI-modfed VSG was protected from protenase K dgeston, ndcatng a lumenal orentaton (compare lanes 2 and 6, denstometrc data are provded n the legend to Fg. 6). We are unable to provde a smple explanaton for the observaton that a fracton of the GPI-modfed VSG (27%) s susceptble to protease (Fg. 6) even though the lumenal marker BP s completely protected n the same experments (Fg. 4). One possblty s that some vescles become unsealed durng mcrosome preparaton. It mght be antcpated that these vescles lose BP and escape detecton n the The Journal of Cell Bology, Volume 127,

7 Fgure 6. GPI-modfcaton of VSG acceptors. GDP-[3H]mannose - labeled membranes (10 s cell eq) were ncubated wth the ndcated concentratons of protenase K for 30 mn on ce. Proteolyss was stopped by addng PMSF and the samples were analyzed by SDS- PAGE on a 10% gel, followed by autoradography oftbe dred gel. Denstometrc analyss gave the followng results (n arbtrary unts): control sample (lane 2, 100 #g/ml protenase K n the presence of PMSF), 25595; protease-treated sample (lane 6, 100 #g/ml protenase K), (73% of control). BP protease protecton assays, but retan the ablty to synthesze and transfer GPI to VSG, gvng rse to GPI-modfed VSG that s accessble to protease dgeston n the absence of detergent. We thnk ths unlkely as BP and other ER retculoplasmns probably exst n two phases n the ER lumen: a moble phase, free to dffuse n the lumenal space and a calcum-stablzed mmoble matrx that s not easly removed by vescle dsrupton (Booth and Koch, 1989). Indeed t s dffcult to obtan quanttatve release of retculoplasmns such as proten dsulfde somerase by smply osmotcally shockng the mcrosomes (Paver et al., 1989), and typcally very lttle BP s lost durng membrane solaton procedures of the type we use (Bangs et al., 1993). The damaged vescles would therefore be expected to retan some BP and contrbute a dagnostc proteolytc fragment n protease assays wthout detergent: no such fragment was detected (Fg. 4, lane 3). An alternate hypothess s that GPI transfer to proten occurs on the cytoplasmc face of the ER and that we detect a fracton of the VSG that s not translocated to the ER lumen after GPI modfcaton. Ths possblty appears unlkely gven current nformaton on ER proten translocaton mechansms (Glmore, 1993). bound. Nevertheless the un-normalzed data ndcate that, wth the excepton of P3, all the n vtro syntheszed GPIs probed are predomnantly (>60%) located n the external leaflet of the mcrosomal vescles (Fg. 7). The results suggest that the GPI transferases and the GIcNAc-PI de-n-acetylase have cytoplasmcally orented actve stes. Indrect data n support of ths proposal have been recently publshed (Mensa-W'dmot et al., 1994). Bloodstream-form trypanosomes synthesze two forms of transfer-competent (phosphoethanolamne-contanng) GPIs, P2, and P3, dfferng only n that P3 s nostolacylated whle P2 s not (Mayor et al., 1990a,b). The two lpds appear to exst n dynamc equlbrum, undergong nterconverson through nostol acylaton and acyl chan hydrolyss (Gther et al., 1994). Although the precse role of nostol acylaton n GPI assembly remans to be defned, t has been suggested that t may provde a crtcal stereochemcal constrant essental for glycan assembly (Menon et al., 1990a) or functon as a tag for GPI reservors or excess GPI pools destned for catabolsm (Gther et al., 1994). Our data show that whle both P3 and P2 are equally recognzed by Con A n detergent soluton and both lpds are accessble to Con A n ntact mcrosomal vescles, sgnfcantly less mcrosomal P3 reacts wth the lectn. We were unable to pursue ths observaton further wth membrane-mpermeant probes drected aganst the amno group of ethanolamne n both lpds. We tested trntrobenzenesulfonc acd and the N-hydroxysuccnmde ester of botn: both reagents mod- Dscusson GPIs Are Syntheszed on the Cytoplasmc Face of the ER The constructon of the GPI anchor precursor s a multstep process nvolvng at least four glycosyltransferases, one nostol acyltransferase, one deacetylase, and one ethanolamnephosphotransferase. The enzymes are located n the endoplasmc retculum but the arrangement of enzyme actve stes and hence the topology of the assembly process has not been descrbed. In ths paper we use two membrane-mpermeant probes, Con A and phosphatdylnostol-specfc phospholpase C, to analyze the transblayer dstrbuton of GPI bosynthetc lpd ntermedates. All the GPIs probed (GlcNAc-PI, GlcN-PI, M1, M2, M3, P2, M3*, and P3) were accessble to these reagents n ntact mcrosomal vescles (data summarzed n Fg. 7). As t s dffcult to assess the effcency wth whch PI-PLC and Con A recognze membrane-bound substrates, the fracton of mcrosomal GPI detected usng these probes necessarly represents a lower Fgure 7. Orentaton of GPI structures n mcrosomal membranes. The fgure summarzes data on the accessblty of mcrosomal GPI structures to varous topologcal probes. The mcrosomal orentaton of newly GPI-modfed VSG was determned by protease treatment (100/~g/ml protenase K, 30 mn on ce). The transblayer dstrbuton of lpd ntermedates was probed wth PI-PLC (1.7 U/rrd, 20 mn, ce) and/or Con A (5 mg/ml, 20 mn, ce) as descrbed n the text. The fracton of each lpd accessble to the probe (dark bars) s not normalzed to probe effcency and should therefore be taken as a lower lmt of the amount of lpd presentn the external (cytoplasmc) leaflet of the mcrosoma membrane. The % naccessble (lght bars) s obtaned by subtracton. Varaton n the data over several experments was <10%. The data are taken from Fg. 1 A (GIcNAc-PI and GIcN-PI), Fg. 2 and the text (M2), Table II (P2 and M3, PI-PLC + Con A), Fg. 5 (P3), text (M1 and M3* PI-PLC/Con A, mcrosomes wthout PMSF), and Fg. 6 (VSG). Vdugrene et al. Glycosylphosphatdylnostol Anchor Assembly 339

8 fed P2 and P3 n organc solvents but not under mlder, physologcally approprate condtons (unpublshed data). Data for another nostol acylated GPI (M3*) ndcate that at least 65 % of ths speces s present on the external face of the mcrosomal vescles, suggestng that nostol acylaton alone s not responsble for the relatve naccessblty of mcrosomal P3. The sgnfcance of the oppostely skewed dstrbutons of P2 and P3 n mcrosomal vescles (Fg. 7) s not clear, but the observaton may be relevant to an understandng of the possble effect on GPI metabolsm of the GPIspecfc phospholpase C expressed n bloodstream trypanosomes and located on the cytoplasmc face of otherwse undefned membrane vescles (Bllow et al., 1989; Mensa- Wlmot et al., 1994). The GPI pathway may be compared to other ER lpd glycosylaton pathways nvolvng dolchol phosphate. The synthess of dolchol-lnked olgosaccharde structures n the ER proceeds n two phases. The ntal seven-sugar structure (MansGlcNAc2-PP-dolchol) s syntheszed on dolchol phosphate from sugar nucleotde donors on the cytoplasmc face of the ER, and then elongated wth sugars (four mannose resdues and three glucose resdues) derved from dolchol-p-man and dolchol-p-glucose donors on the lumenal face of the ER (Abejon and Hrschberg, 1992). In ths reacton sequence, as n yeast O-mannosylaton (Tanner and Lehle, 1987), dolchol-p-mannose-dependent glycosylaton reactons occur on the lumenal face of the ER. Thus, despte the many ponts of smlarty between GPI assembly and other ER glycosylaton reactons, the results descrbed n ths paper suggest that GPI bosynthess unquely nvolves the use of a lpd-lnked sugar (dolchol-p-mannose) by a cytoplasmcally orented eukaryotc glycosyltransferase. Newly GPl-modfed VSG Is Manly Located n the Lumenal Leaflet of the ER Despte the now consderable body of nformaton on GPI assembly, the enzymology of proten modfcaton by GPI s poorly understood. It has been generally assumed that a pseudotranspeptdaton (transamdaton) reacton s nvolved, and that the cleavage of the proten carboxyl-termnal GPIdrectng sgnal sequence and attachment of GPI occurs n concerted fashon wthout external energy nputs. Snce GPI modfcaton occurs n the absence of ongong proten translaton (Mayor et al., 1991; Amthauer et al., 1992) and the GPI-drectng sgnal sequence s at the carboxyl termnus of the proten (Cross, 1990), t s lkely that GPI attachment occurs after proten translocaton across the ER membrane, possbly after release of the polypeptde from the translocaton apparatus. We nvestgated the GPI addton reacton by explotng the fact that trypanosome mcrosomes contan at least 4,000 copes of unprocessed VSG per cell equvalent (Mayor et al., 1991) that are avalable for modfcaton by n vtro syntheszed GPIs. Our analyses of ths sytem va protease protecton experments confrm that newly GPIanchored VSG molecules are prmarly lumenally orented, consstent wth ther eventual expresson at the cell surface. These results do not drectly address the ssue of the topology of GPI transfer to proten, and the statement that transfer occurs n the lumenal leaflet must be regarded as smply a possblty, albet a lkely one, untl further data are obtaned. Transblayer Movement of Glycolpds In the dolchol pathway of N-glycosylaton, MansGlcNAc2- PP-dolchol s located predomnantly n the cytoplasmc leaflet of the ER whle Man6GlcNAc2-PP-dolchol and more elaborate speces are located prmarly n the lumenal leaflet (Snder and Rogers, 1984). These data are best accounted for by the proposal that a certan fracton of MansGlcNAc2-PPdolchol flps nto the lumenal leaflet of the ER and s rapdly elaborated to Man~GlcNAc2-PP-dolchol. The synthess of ganglosdes n the Golg may also nvolve flppng of a partally glycosylated lpd precursor, snce the bosynthetc reactons nvolve synthess of glucosylceramde (and possbly lactosylceramde) on the cytoplasmc face and subsequent processng on the lumenal face (van Meet, 1993). The data presented n ths paper suggest that the GPI endproducts, P2 and P3, may undergo smlar transblayer movement. Thus expermental characterzatons of lpd glycosylaton n the ER and Golg are best explaned by postulatng transblayer movement (flp-flop) of glycosylated lpds. In the dolchol-p and PI glycosylaton pathways, lpd speces wth headgroups of consderable polarty must be translocared across the ER membrane blayer. It has been clearly establshed that transport of phospholpds across membrane blayers does not occur spontaneously, and that n some bologcal membranes, partcularly those possessng lpd bosynthetc capablty, transport s facltated by proten catalysts termed flppases or lpd translocases. Studes wth glycerophospholpd substrates have demonstrated several characterstcs of such an actvty (Zachowsk, 1993), but no flppase has been solated. It remans to be seen whether specfc flppases exst for these varous substrates, or whether speces that are flpped are those that are avalable for transport because they have relatvely long half lves or because they are not nvolved n stable lpd-lpd or lpd-proten nteractons. It s concevable that partally assembled GPI structures may not be avalable to flppases (but nevertheless accessble to topologcal probes) because they may be bound to one of the GPI bosynthetc transferases. The expermental work descrbed n ths paper was carred out n The Rockefeller Unversty's Laboratory of Molecular Parastology, and we are grateful to the head of the laboratory, Professor George A.M. Cross for hs support. We thank Adam Stenberg and Laura van der Ploeg for producng the fgures, Jay Bangs for antbodes to trypanosome BP, and Martn Low for PI-PLC. We are grateful to Clauda Abejon, Jay Bangs, Jtu Mayor, Dave Nelson, Chrs Ncchtta, and one of the Journal of Cell Bology revewers for crtcal comments on the manuscrpt, and I. Menon, K. M. M., Ian Rchardson, and Bob Dylan for stmulaton. Ths work was supported by Natonal Insttutes of Health grant AI28858, the Irma T. Hrschl Trust (New York), and the Unversty of Wsconsn. Receved for publcaton 16 June 1994 and n revsed form 27 July References AImjon, C., and C. B. Hrschberg Topography of glycosylaton reactons n the endoplasmc retculum. Trends Bochem. Sc. 17: Amthauer, R., K. Kodukula, L. Brnk, and S. Udenfrend Phosphatdylnostol-glycan (PI-G)-anchored membrane protens: requrement of ATP and GTP for translaton-ndependent COOH-termnal processng. Proc. Natl. Acad. Sc. USA. 89: Amthauer, R., K. Kodukula, L. Gerber, and S. 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