Proc. Nati. Acad. Sci. USAVol. 89, pp. 4319-4323, May 1992Cell Biology

Golgi retention of a trans-Golgi membrane protein, galactosyl-transferase, requires cysteine and histidine residues withinthe membrane-anchoring domain

(glycosyltransferase/glycosylation/mutagenesis/intraceilular targeting transport/oligomerization)

DAISUKE AOKI*, Ni LEEt, NAOTO YAMAGUCHI, CRAIG DUBOISt, AND MICHIKo N. FUKUDA§La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037

Communicated by Stuart Kornfeld, January 23, 1992

ABSTRACT Galactosyltransferase (GT; UDPgalactose:3-D-N-acetylglucosaminide g-1,4-galactosyltransferase, EC2.4.1.22) is a type II membrane-anchored protein composed ofa short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a largecatalytic domain including the C terminus. To identify thepeptide segment and key amino acid residues that are critical forGolgi localization of GT, the expression vector pGThCG wasdesigned to encode the entire GT molecule fused to the C-ter-minal region of human chorionic gonadotropin a subunit(hCGa) as a reporter. COS-1 cells transfected with pGT-hCGexpressed the chimera in the Golgi region, as detected byimmunofluorescence microscopy using anti-hCG antibodies.Two deletion mutants, A tail and A stem, which are lacking mostofthe N-terminal cytoplasmic tail or 10 amino acids immediatelyafter the membrane-anchoring domain, were localized in theGolgi. Replacement mutations of the membrane-anchoring do-main of GT showed that the second quarter of the transmem-brane domain or Cys29-Ala3O-Leu3l-His32-Leu33 is necessary forGT to be retained in the Grolgi. Furthermore, the point mutantsCys29 -* Ser2' and His32 - Leu32 were partially transported tothe plasma membrane, whereas an MaC-Leu31 -- Phe"-Gly3lmutant was localized in the Golgi. Finally, a double mutant,Cys29/His32 -* Ser29/Leu32, was found to be transported effi-ciently to the plasma membrane. The signal-anchoring domainof the transferrin receptor, a type II plasma membrane protein,was then replaced by portions of the GT transmembrane do-main. Although the Cys-Xaa-Xaa-His sequence by itself cannotretain the transferrin receptor in the Golgi, the cytoplasmic halfof the transmembrane domain of GT was partially capable ofretaining the transferrin receptor in the Golgi. These resultssuggest that the cytoplasmic (or N-terminal) half of the trans-membrane domain of GT contributes to the Golgi retentionsignal and that particularly Cys29 and His32 in this region arecritical for GT to be retained in the Golgi.

Eukaryotic cells contain subcellular organelles in whichspecific proteins reside. Defining the mechanisms that causetargeting and sorting of particular proteins to the organellesis essential to understand how subcellular organelles areformed. Although studies on resident endoplasmic reticulum(ER) and lysosome proteins have progressed recently (1, 2),information on the characteristic localization of Golgi resi-dent proteins has been quite limited. Glycosyltransferases, asindicated by their cDNA sequences, are all type II membraneproteins (3). Thus, each of these proteins is composed of ashort N-terminal cytoplasmic tail followed by an uncleavablesignal membrane anchor, a presumptive stem region, and alarge catalytic domain including the C terminus. Each ofthese glycosyltransferases is localized specifically in one of

the cis-, medial-, or trans-Golgi compartments or in thetrans-Golgi network (4).

Subcellular localization of galactosyltransferase (GT;UDPgalactose:f3-D-N-acetylglucosaminide f3-1,4-galactosyl-transferase, EC 2.4.1.22) has been examined by immunoelec-tron microscopy, and the localization of this enzyme in thetrans-Golgi cisternae is well documented (5). The solubleform of GT is derived from the membrane-bound form byproteolytic cleavage at the stem region (6, 7) and subsequentrelease of the catalytic domain (12). Because the catalyticdomain by itself does not stay in the Golgi and is secretedfrom the cells, it is likely that the Golgi retention signal liessomewhere in the region composed of the tail, membrane-anchoring domain, and stem region.

In the present study, we have introduced a series ofdeletion mutations and replacement mutations into the tail,membrane anchor, and stem region of the GT and found thatCys29 and His32 within the membrane-anchoring domain arethe most critical amino acids for GT to be retained in theGolgi.

MATERIALS AND METHODS

Expression Vector and cDNAs. The eukaryotic expressionvector pcDNAI, which is a modified form of the pCDM8vector (8), was obtained from Invitrogen (La Jolla, CA). ThecDNAs for human GT were isolated as reported (6). ThecDNA for human chorionic gonadotropin a chain (hCGa) (9)was kindly provided by J. Rose (Yale University, NewHaven, CT). The cDNA for human transferrin receptor (10)and monoclonal anti-transferrin receptor antibodies (11) werekindly provided by I. Trowbridge (The Salk Institute, LaJolla, CA).

Plasmid Construction and Mutagenesis. GT cDNA encod-ing the full-length coding region for GT was fused to the XbaI/BamHI hCGa fragment (350 base pairs) encoding theC-terminal half of hCGa in-frame. The cDNA encodingfull-length GT and the hCG chimera was ligated in pcDNAIvector (8) at the EcoRI and Xho I sites. The resulting pcDNAIplasmid is designated as pGT-hCG. Site-specific mutagenesisfor all deletion mutants, replacement mutants, and pointmutants was performed as described (12).The cDNA for human transferrin receptor was inserted at

HindIII and EcoRI sites of pcDNAI. Deletion of the cyto-

Abbreviations: GT, galactosyltransferase; hCG, human chorionicgonadotropin; FITC, fluorescein isothiocyanate; ER, endoplasmicreticulum.*Present address: Department of Gynecology and Obstetrics, KeioUniversity School of Medicine, Tokyo, Japan.

tPresent address: Department of Immunology, Fred HutchinsonCancer Center, Seattle, WA 98104.tPresent address: Pre-Medicine Program, Johns Hopkins Univer-sity, Baltimore, MD 21205.§To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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plasmic tail from the transferrin receptor was achieved byPCR (13) using oligonucleotides TRGTnIAl and TRReV asprimer and TRGTm mutant cDNA as template.

Transfection of COS-1 Cels and Immunofluorescence Mi-croscopy. Transfection of COS-1 cells was carried out by theDEAE-dextran method (14). After culturing for 48 hr, thecells on coverslips were fixed with 1% paraformaldehyde for15 min and then treated with phosphate-buffered saline (PBS)containing 1% bovine serum albumin with or without 0.1%saponin (15). The cells were incubated with anti-hCG anti-serum at room temperature for 1 hr. After washing with PBS,cells were stained with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Fab')2 and examined by immu-nofluorescence microscopy. Immunofluorescence for COS-1cells expressing transferrin receptor was performed by usinga monoclonal anti-transferrin receptor antibody and FITC-labeled rabbit anti-mouse IgG (Fab')2 fragment.Flow Cytometry. The COS-1 cells were harvested from the

tissue culture plate by incubation in enzyme-free cell disso-ciation solution (Specialty Media, Lavallette, NJ). The cellswere incubated with rabbit anti-hCG antibodies on ice for 20min, followed by incubation with FITC-conjugated goatanti-rabbit IgG (Fab')2 fragment on ice for 20 min. The cellswere suspended in the cell dissociation solution containingpropidium iodide (10 ,kg/ml) before being subjected to anal-ysis with a FACScan (Becton Dickinson).

Immunoprecipitation. The COS-1 cells transfected withexpression vector plasmid were metabolically labeled with[35S]methionine plus [35S]cysteine in methionine-free RPMImedium, supplemented with Tran35S-label (ICN Radiochem-icals) (100 ,Ci/ml; 1 Ci = 37 GBq) for 1 hr, and chased withcomplete medium for 2 hr. Cell lysates were prepared with1% Nonidet P-40. Immunoprecipitation was performed withanti-hCG antibodies (Cappel Laboratories) as described (16),except protein G Sepharose (Pharmacia) was used in place ofStaphylococcus aureus cells.Assay of GT Activity. The COS-1 cells transfected with

pcDNAI vector alone or pGT-hCG plasmid were harvestedwith rubber policemen. The crude homogenates were pre-pared as described (17). Enzyme activity ofGT was assayedas described (12) with [3H]UDPgalactose and di-N-acetylchitobiose.

RESULTSExpression of GT-hCG Chimera in COS-1 Cells. GT is

expressed in all mammalian cells. In our studies, it is impor-tant to be able to distinguish between exogenous human GTand the endogenous monkey GT expressed in COS-1 cells.We therefore chose to tag GT with hCG peptide. Thefull-length human GT peptide was fused to the 59 C-terminalamino acids of the hCGa peptide (Fig. 1). The fusion proteincan readily be detected by anti-hCG antibodies, since hCG,a peptide hormone, is not expressed naturally in COS-1 cells.The cDNA coding for this fusion protein was inserted into theeukaryotic expression vector pcDNAI. COS-1 cells weretransfected with this plasmid DNA and expression of thefusion protein was examined as follows.Crude homogenates of COS-1 cells were assayed for GT

activity. The homogenate prepared from the transfectedCOS-1 cells with pGT hCG exhibited a GT activity of 35.9 x10-3 units per mg of protein, while the homogenate preparedfrom the transfected COS-1 cells with vector alone showed aGT activity of 9.9 x 10-3 units per mg of protein. As thetransfection efficiency in our experiments was 20-30%, thisdegree (3.6-fold) of overexpression is similar to the previ-ously reported activity levels seen for COS cells transfectedwith GT cDNA (18, 19). The transfected COS-1 cells weremetabolically labeled and the immunoprecipitates with anti-hCG antibodies from the total cell lysate were examined by

[ iWIZ] - -4 4

Deletion Mutants (tail and stem region)

A Tall

A Stem

5 23

MRLR EPLLSGAAMPGASLORACR4445 54 77R )LSRLPQLVG VSTPLQGGSNSAAAI GQSSGELR

Replacement Mutants and Point Mutants( membrane anchoring domain )

29 32wild type L L V A V C A L H L G V T L V Y Y L A G

TRm I

TRm2

TRm3

TRm4

VICYGT ----------------

- - - - - A V V F - - - - - - - - - -

- - - - - - - - - - - F L G FM.-- - - -

---------------- GYLGY

29

30 31A L-4FG - - - - - - -F G -- - - - - - - - - - - -

32H -L ---------L-------------29 32

C H-SL - - - - - -SS-- L - - - - - - - - - - --

FIG. 1. Schematic diagram ofwild-type GT-hCG chimeric proteinand various mutants ofGT hCG used in this study. The tail and stemregion was examined by deletion mutations. ATail mutant lacks 19amino acids (residues 5-23; boxed) in the 23-amino acid N-terminalcytoplasmic tail. AStem mutant lacks 10 amino acids (residues 45-54;boxed) immediately after the membrane-anchoring domain. Themembrane-anchoring domain was examined by replacement muta-tions and point mutations. In TRml, -2, -3, and -4, all five amino acidsin the membrane-anchoring domain of GT were replaced with thecorresponding six amino acids ofthe membrane-anchoring domain ofthe transferrin receptor.

SDS/PAGE followed by fluorography (Fig. 2). Two bandswith apparent molecular masses of 63 and 56 kDa weredetected as specific immunoprecipitates. Earlier studies onnative GT in various types of cells also showed two bands,and the upper and lower bands were characterized as amature form GT with both N- and O-glycans and a precursorform GT with N- but not O-glycans, respectively (20). Theseresults indicate that upon transfection of pGT hCG plasmid,COS-1 cells express enzymatically active and immunologi-cally recognizable GT-hCG chimera molecules.

Subcellular localization of the GT-hCG chimera was ex-amined by immunofluorescence microscopy. In COS-1 cells,GT localization was restricted to a crescent-shaped reticularpattern adjacent to the nucleus (Fig. 3A). This pattern ofimmunofluorescence is characteristic for the Golgi complexand is seen with native GT (5, 21). Mock transfections, ortransfections with vector alone, resulted in no detectableimmunofluorescence signal (data not shown).About 20o of transfected COS-1 cells apparently ex-

pressed a high level of GT-hCG, and such cells exhibited animmunofluorescence signal not only in the Golgi region butthroughout the cytoplasm in a reticular structure character-istic ofthe ER. Similar results have been reported (17, 22, 23)for expression of Golgi proteins. These observations suggest

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1 2

200-

116-97-

66-

43-

31-

22-

FIG. 2. Immunoprecipitation ofGT-hCGfusion protein. COS-1 cells transfected with

m* ~. cDNA encoding wild-type GT-hCG weremetabolically labeled with [35S]methionine.Immunoprecipitates were analyzed by SDS/PAGE followed by fluorography. Lanes: 1,immunoprecipitates with normal rabbit se-rum; 2, immunoprecipitates with anti-hCGantiserum. Arrows show specific immuno-precipitates with anti-hCG antibodies. Num-bers (kDa) on the left side show migrationpositions of molecular mass markers.

that the high level of expression of these Golgi proteinsresults in their detection in the ER.As shown in Fig. 3, immunofluorescence of the fusion

protein GT-hCG in COS-1 cells partially overlaps with thearea stained with wheat germ agglutinin. This lectin, whichbinds to the glycoconjugates having N-acetylglucosamineand sialic acid, has often been used as a Golgi marker reagent,but this lectin can also detect lysosomes and plasma mem-brane (24). Immunofluorescence with the GT-hCG, however,did not reveal staining of the lysosomes and plasma mem-brane (Fig. 3). These results indicate that recombinantGT-hCG expressed in COS-1 cells is mostly localized in theGolgi, where the GT naturally resides.

Identification of Peptide Segment Necessary for Golgi Re-tention. As the first step toward finding the peptide segmentinvolved in the Golgi retention of GT, two deletion mutants,A tail and A stem, were made (see Fig. 1). The A tail mutanthas a deletion from Glu5 to Arg23 and thus lacks most of thecytoplasmic tail except for Met'-Arg2-Leu3-Arg4. The A stemmutant has a deletion from Asp45 to Gly54 and thus lacks 10amino acids adjacent to the membrane-anchoring domain inthe stem region. The subcellular localization of these mutantproteins in the COS-1 cells was examined by immunofluo-rescence microscopy. As shown in Fig. 4, both A tail and Astem mutants localized in the Golgi (Fig. 4I A and B). Theresults suggest that Golgi retention of GT is not criticallyaffected by these deleted peptide segments.

FIG. 3. Immunofluorescence ofCOS-1 cells expressing wild-typeGThCG chimera. (A) COS-1 cells were transfected with pGThCGvector. The cells were fixed and permeabilized with saponin. TheGT-hCG chimeric protein was detected with anti-hCG antibodies andFITC-conjugated goat anti-rabbit IgG. (B) Immunofluorescence ofCOS-1 cells with rhodamine-conjugated wheat germ agglutinin. Thesame field of COS-1 cells as in A is shown.

The membrane-anchoring domain of GT was then exam-ined by replacement mutations (see Fig. 1). This domain,which consists of 20 amino acids, was divided into fourblocks. Each block ofthe GT membrane anchor was replacedwith 6 amino acids from the corresponding membrane-anchoring domain of the transferrin receptor. The transferrinreceptor was chosen because it is also a type II membrane-anchoring protein (10) and is transported to the plasmamembrane. Thus, the transferrin receptor is assumed not tohave a Golgi retention signal.Immunofluorescence of the TRml mutant showed tight jux-

tanuclear staining (Fig. 4I C) but never showed crescent-shapedreticular distribution, a pattern that was characteristically ob-served in the immunofluorescence staining of wild-typeGT-hCG. This suggests that the TRml mutant localizes in arestricted region of the Golgi but not trans-Golgi. It is alsopossible that the TRml mutant is accumulated in the so-calledRussell bodies, organelles accumulating truncated proteins thatcan neither exit from, nor be degraded in, the ER (25).

Localization of the TRm2 mutant showed that this mutantwas transported to the plasma membrane (Fig. 4I D). Strongto moderate surface expression of the TRm2 mutant wasdetected by flow-cytometry analysis (Fig. 5B). On the otherhand, both the TRm3 and TRm4 mutants localized in theGolgi (Fig. 4IE and F). These results suggest that the secondblock of the GT membrane-anchoring domain composed ofCys29-Ala30-Leu31-His32-Leu33 plays a critical role in Golgiretention of GT.

11

FIG. 4. Immunofluorescence ofCOS-1 cells transfected with pGT-hCG mutant DNAs. (I) Localization ofeach deletion mutant and replacementmutant protein. (A) A tail. (B) A stem. (C) TRml. (D) TRm2. (E) TRm3. (F) TRm4. (II) Localization of each point-mutant protein. (A) Cys29 -SerP. (B) Ala3Q-Leu31 -> Phe3l-Gly31. (C) His32 -- Leu32. (D) Cys29/His32 -. Ser2/Leu32. In both I and II, the cells were permeabilized beforebeing subjected to immunostaining.

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102 103Fluorescent Intensity (log)

FIG. 5. Flow cytometryanalysis of cell-surface expres-sion ofGT hCG mutant proteinson COS-1 cells. (A) Wild-typeGThCG. (B) TRm2 mutant. (C)Cys2- Ser" mutant. (D) Alai-Leu31 Phe3Q-Gly31 mutant. (E)His32 Leu32 mutant. (F) C29/H32 -. Ser29/Leu32 double mu-tant. The proportion of cellsgated as positive for each cellpopulation (horizontal bars) was9%oin A, 33% in B, 12% in C, 6%in D, 17% in E, and 24% in F.

Identification ofAmino Acids Necessary for Gogi Retention.To define further which particular residues in the region fromCys9 to Leu32 are important for Golgi retention, pointmutants were constructed (see Fig. 1). As shown in Fig. 4, adouble point mutation, AlaW-Leu3l -- Phe3.-Gly"', had no

effect on Golgi localization (Fig. 411 B). Significantly, how-ever, the point mutants Cys29- Ser" and His32 -_ Leu32

showed diffuse Golgi staining (Fig. 411 A and C). Weak butdefinite surface expression was detected by immunofluores-cence microscopy (data not shown) as well as by flowcytometry (Fig. 5 Cand E). However, the Cys29- SerP and

His32 -+ Leu32 mutations still permitted some of the mutant

protein to be retained in the Golgi (Fig. 411A and C). Finally,a double-mutant Cys29/His32 +Sei9/Leu32 was con-

structed. The staining pattern ofthis mutant was quite similarto that observed with the TRm2 mutant (Fig. 41 E) and wastransported efficiently to the cell surface. These resultsindicate that efficient retention ofGT in the Golgi depends onthe presence of both Cys9 and His32 residues.A Potential Golgi Retention Signsl of the Transmembrane

Domain Containing Cys' and His2. As described above, ourresults indicated that the cytoplasmic (or N-terminal) half ofthe transmembrane domain of GT is necessary for Golgitargeting and retention. We therefore tested whether thisregion is capable of retaining any type II protein, such astransferrin receptor, in the Golgi.The COS-1 cells expressing cDNA encoding wild-type

transferrin receptor gave a strong surface staining (Fig. 61IA). A transferrin receptor mutant, TRCH, in which Val" andPhe76 were replaced by Cys73 and His76, was transported tothe cell surface (Fig. 611 B), indicating that the mere intro-duction of cysteine and histidine in the transmembranedomain does not create a Golgi retention signal. We con-structed a transferrin receptor mutant, TROTm, in which thecytoplasmic 10 amino acids of the membrane anchor oftransferrin receptor were replaced with 10 amino acids of thecytoplasmic half of the membrine-anchoring domain of GT.The TRGTm mutant was, however, largely transported to theplasma membranes (Fig. 6II C). Considering that the cyto-plasmic tail oftransferrin receptor may hinder Golgi retentionin this particular case, the TRGTm mutant was modified toTRGTmL W mutant by deleting the cytoplasmic tail of thetransferrin receptor. The immunofluorescence of theTRGTmS mutant in the Golgi was significantly increasedcompared to wild type (Fig. 61I D). This result suggests thatthe cytoplasmic half of the transmembrane domain of GT ispartially capable of functioning as the Golgi retention signal.

DISCUSSION

The present study was ainie,' at identifying the peptidesegment and the key amino acid residues responsible for

Golgi retention of GT. The results obtained by deletion,replacement, and point-mutation experiments (Figs. 1, 4, and5) indicate that the cytoplasmic side of the membrane-anchoring domain, particularly Cys29-Xaa30-Xaa31-His32,plays a critical role for GT to be retained in the Golgi.Observations ofTRml and TRm2 mutants (Figs. 4 and 5) ledus to focus on the cytoplasmic (or N-terminal) half of thetransmembrane domain. A dramatic effect of the TRm2replacement mutation and point mutations within the secondquarter of the transmembrane domain indicates that thisregion is most important for Golgi retention of GT. It may

cytoplasm ic

tail

Wild

membraneanchoringdomain

lumenaldomain

- - - -i SICYGTIAV IVFFLIGFMIGYLGY -

TRCH ---- - I - - - -'- C--H- - - - - - - - - - -

TR-Tm- - - - -I LLVAVCALHL-

TRGTmAta MAKR LLVAVCALHL-

FiG. 6. Localization of transferrin receptor and its mutants. (I)Schematic diagram of wild-type transfernn receptor and mutants oftransferrin receptor used in this study. In TRCH mutant, Val73 andPhe76 in the transmembrane domain have been changed to Cys3 andHis , respectively. In TRGTm mutant, the cytoplasmic half of thetransferrin receptor membrane-anchoring domain has been replacedwith 10 amino acids from the cytopla*mic half of the membrane-anchoring domain of GT. In the TRRGTml mutant, 60 amino acidsofthe cytoplasmic tail have been deleted from the TRGTm mutant. (1I)Immunofluorescence of COS-1 cells transfected with cDNAs encod-ing transferrin receptor and its mutant. (A) Wild type. (B) TRCHmutant. (C) TRGTm mutant. (D) TRGTDtaIt mutant.

.0Ez

0=

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also be possible that replacement mutations such as TRm2changed the conformation of the other part of the transmem-brane domain, resulting in release of GT from the Golgi.However, mutation of Cys29 to Ser2 should cause a minimalchange of peptide conformation; yet, it clearly resulted inrelease of GT from the Golgi. We have also constructed theHis32 -> Phe32 and the His32--+ Arg32 mutant. These mutantswere not retained in the Golgi and gave a staining patternsimilar to that of the His32 -- Leu32 mutant (data not shown).These observations together with the results obtained withCys29 -- Ser9, His32 -- Leu32, and Cys29/His32 _) Ser2/Leu32 mutants favor the conclusion that Cys9 and His32residues are directly involved in Golgi retention of GT. Theunusual localization of the TRml mutant (Fig. 4C) suggeststhat this region is also necessary for proper targeting of GTto the Golgi apparatus. Further analysis is needed to definethe role of this region.

In contrast to TRml and TRm2 mutations, we did notdetect obvious effects ofmutating the lumenal (or C-terminal)half of the transmembrane domain (see TRm3 and TRm4mutations; Fig. 4). We focused on the tyrosine residuespresent in this region because tyrosine is known to play acritical role in endocytosis (2) and because in other glyco-syltransferases the aromatic amino acids are present in thelumenal side of the transmembrane domain. Point mutationssuch as Tyr39 -* Ala39 and Tyr39-Tyr40 -Aa39-lea0, how-ever, did not alter Golgi localization of GT (data not shown).While this manuscript was in preparation, Nilsson et al.

(22) reported the Golgi retention signal of GT. They showedthat the lumenal half of the membrane-anchoring domain ofGT can retain the GT-invariant chain chimera in Golgi (22).On the other hand, the present study indicates that thecytoplasmic half of the transmembrane domain of GT alsocan function as a Golgi retention signal in a dominant manner(Fig. 6). In addition, the presence of the Golgi retention signalwithin a membrane-anchoring domain has been shown foravian coronavirus El glycoprotein, which is retained in thecis-Golgi. An important feature of the Golgi retention signaldescribed was the uncharged polar residues that line one faceof an a-helix (26). As the predicted conformation of thecytoplasmic half of the transmembrane domain of GT isa-helix, Cys29 and His32 are likely to face the same side of thehelix. This structure could be recognized by other protein(s)in the membranes, and such protein-protein interactionscould be responsible for targeting and retention of GT in theGolgi.The drastic effect ofthe mutation from Cys29 to Ser29 raised

the question of whether Cys29 is involved in disulfide cross-linking. Earlier studies on native GT in HeLa cells andhepatoma cells provided no sign of di- or polymerization ofGT in the Golgi membranes (20). The molecular mass ofwild-type GT-hCG and TRm2 mutant proteins in nonreducinggels showed that the monomeric form is the major species forboth wild-type GT-hCG and TRm2 mutant proteins (unpub-lished results). Therefore, intermolecular disulfide cross-linking, if it occurs at Cys29, is not a necessary factor for Golgiretention.The role of Cys29 and His32 for Golgi retention might be

dependent on their unique structures. It is noteworthy thatcysteine and histidine are often involved in metal bindingsites. In zinc finger proteins, cysteines and histidines com-pose the zinc binding peptide motif (27). In cytochromeoxidases, these two amino acids bind iron and copper (28). Arecently identified yeast zinc finger protein, Vpsl8p, plays arole in protein sorting in the late Golgi (29). Furthermore, thepossibility for a zinc binding cation activator in the Golgivesicle was predicted by kinetic analysis of GT in the Golgi(30). It is therefore an interesting possibility that GT mole-cules could interact with each other or bind other molecule(s)at Cys29 and His32 through metal ions such as zinc.

A possible mechanism for Golgi retention would be if thetransmembrane regions of GTs recognize each other to formoligomers, which become large enough to immobilize them-selves, as suggested for ER proteins (31). Oligomerizationmust be facilitated in the specific environments of the trans-Golgi, where some endogenous protein and/or lipid couldpromote oligomerization of the GT under appropriate pHand/or ionic strength conditions. It also seems possible thatthe GT dimer results in creation of an epitope that is recog-nized by an endogenous protein, which functions as a recy-cling receptor, the mechanism analogous to ER residentproteins retained by the Lys-Asp-Glu-Leu receptor (1, 32).The expression vector for the GT-hCG fusion protein and aseries of mutants obtained in this study will be useful fortesting these possible Golgi retention mechanisms.

We would like to thank Dr. Mark Williams for his helpful sugges-tions and critical discussions. We thank Dr. Donald Newmeyer forhis pertinent advice on the manuscript, Allison Hughes for herexcellent technical help, and Henny Bierhuizen for secretarial as-sistance. This work has been supported by a grant (RO1-DK37016)from the National Institutes of Health. N.Y. is a recipient ofa UeharaMemorial Foundation research fellowship.

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