©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Golgi Retention Mechanism of -1,4-Galactosyltransferase
MEMBRANE-SPANNING DOMAIN-DEPENDENT HOMODIMERIZATION AND ASSOCIATION WITH - AND -TUBULINS (*)

Naoto Yamaguchi (§) , Michiko N. Fukuda (¶)

From the (1) La Jolla Cancer Research Foundation, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent studies on proteins residing in the Golgi complex revealed that the membrane-spanning domain of these proteins are largely responsible for their retention in the Golgi complex. We show here that -1,4-galactosyltransferase (GT) forms homodimers and large oligomers in vivo, and the formation of the homodimers is dependent on cysteine and histidine residues within the transmembrane domain. Double mutations of these residues, Cys Ser and His Leu, abolish homodimerization and simultaneously reduce the Golgi retention. Co-immunoprecipitation of GT and various GT chimeras with anti-GT and anti-reporter molecule antibodies revealed that large aggregates of GT are associated with - and -tubulins and also with other cellular proteins. This association between tubulins and GT suggests a supportive role of the cytoskeleton in the Golgi retention mechanism.


INTRODUCTION

Newly synthesized proteins are either released into the cytoplasm or, if they contain a signal peptide, are translocated into the ER,() entering into the sorting pathway (Walter and Lingappa, 1986). Intracellular transport of proteins from the ER to plasma membranes is mediated by a series of carrier vesicles that bud from one compartment and then fuse with the next, giving rise to vectorial movement (Rothman and Orci, 1992). This transport is believed to occur by ``default,'' meaning that proteins which do not have either a signal for transport to a particular organelle or a specific retention signal are transported to plasma membranes (Pfeffer and Rothman, 1987).

The Golgi complex plays a central role in intracellular sorting and transport (Palade, 1975). The Golgi complex is also the site glycosylation of nascent proteins takes place; there are over 100 different glycosyltransferases involved in the synthesis of protein- and lipid-bound oligosaccharides (Schachter and Roseman, 1980; Beyer and Hill, 1982). While newly synthesized proteins pass through the Golgi, these enzymes are retained in the Golgi complex against membrane ``bulk flow.'' The mechanism underlying such an apparent immobilization of glycosyltransferases in the Golgi membranes has not been well understood.

Galactosyltransferase (GT; UDP-galactose:-D-N-acetylglucosaminide -1,4-galactosyltransferase, EC 2.4.1.22) is one of the best known glycosyltransferases and catalyzes the transfer of galactose from UDP-galactose to N-acetylglucosamine, forming the Gal-1,4-GlcNAc linkage present in glycoproteins, glycolipids and proteoglycans (Beyer and Hill, 1982). The biosynthesis of GT showed that GT is retained in the Golgi with a half-life for 21 h and is also transported to plasma membranes in HeLa cells (Strous and Berger, 1982; Strous, 1986). The localization of GT in the trans-cisternae of the Golgi complex has been well documented by immunoelectron microscopy (Roth and Berger, 1982). In some types of cells, GT is also present in plasma membranes (Lopez et al., 1985; Roth et al., 1985; Bayna et al., 1988; Suganuma et al., 1991). The association of GT with the cytoskeleton has been suggested in some reports (Eckstein and Shur, 1992; Strous et al., 1991), although the significance of such observations in relation to the Golgi retention has not been explored.

GT is a type II integral membrane protein (Masri et al., 1988; Shaper et al., 1988; Nakazawa et al., 1988; D'Agostaro et al., 1989), as are all the Golgi glycosyltransferases cloned to date; they contain a short amino-terminal cytoplasmic tail, a non-cleavable signal-anchor/transmembrane domain, a lumenal stem region, and a large carboxyl-terminal catalytic domain exposed to the Golgi lumen (Paulson and Colley, 1989).

Recent studies have shown that sequences within and adjacent to the transmembrane domain of these Golgi enzymes specify Golgi retention (Munro, 1991; Nilsson et al., 1991; Aoki et al., 1992; Burke et al., 1992; Colley et al., 1992; Russo et al., 1992; Tang et al., 1992; Teasdale et al., 1992; Wong et al., 1992). Importance of membrane-spanning domains for Golgi retention has been shown in virus envelope proteins which are also retained in the Golgi (Machamer and Rose, 1987; Swift and Machamer, 1991). There is, however, no amino acid sequence similarities in the transmembrane domain among these Golgi proteins. Because of the lack of a common amino acid sequence within and/or adjacent to the transmembrane domain in these Golgi proteins, it is difficult to predict a peptide sequence for a Golgi retention signal. This is in contrast to that of ER resident proteins, which are distinguished by the presence of a carboxyl-terminal tetrapeptide. Proteins bearing this signal can be retrieved from the Golgi complex back to the ER by the receptor that recognizes this signal motif (Lewis and Pelham, 1992). It is not known, however, if the proteins residing in the Golgi are recycled in a similar retrieving machinery.

In this report, we show data suggesting that GTs are forming homodimers in vivo, and such homodimerization is largely dependent on Cys and His residues in the transmembrane domain. Mutation of these two amino acid residues significantly affect the retention of GT in the Golgi complex. Furthermore, we show that - and -tubulins are associated with oligomers of GT, suggesting that such an interaction may be an important factor for Golgi retention mechanism.


MATERIALS AND METHODS

Chimeric Constructs

cDNAs encoding human CG (Fiddes and Goodman, 1979) and TfR (Schneider et al., 1984) were kindly provided by Drs. J. Rose (Yale University, New Haven, CT) and I. Trowbridge (The Salk Institute, La Jolla, CA), respectively. cDNAs encoding human GT (Masri et al., 1988), double-point mutated GT, GT tagged with the C-terminal half of human CG, and human TfR inserted into the eukaryotic expression vector pcDNAI (Invitrogen, San Diego, CA) have been described previously (Aoki et al., 1990, 1992). Introduction of RsrII and XhoI sites into the CG cDNA generated a fragment of the cDNA encoding the mature CG protein lacking a signal sequence. This fragment was ligated at the RsrII and XhoI sites in pcDNAI containing GT and to a corresponding fragment containing double-point mutated GT cDNAs, resulting in GTCG and GTCG, respectively (see Fig. 1). GTTfR and GTTfR were constructed in the same manner as GTCG by introducing RsrII and XbaI sites into the extracellular domain of TfR (amino acids 97-760).


Figure 1: Schematic diagram of human GT and TfR and their chimeric proteins. SL mutants represent a double point mutation in the GT transmembrane domain, Cys Ser and His Leu. CG, human chorionic gonadotropin subunit (Fiddes and Goodman, 1979); C, cysteine; H, histidine; S, serine; L, leucine; CYT, cytoplasmic tail; TM, transmembrane domain; STEM, stem region; EX, extracellular region; CATALYTIC, catalytic domain. The numbers refer to the amino acid sequence in the wild-type protein.



Antibodies

Rabbit anti-human CG antibodies, fluorescein isothiocyanate-conjugated F(ab`) fragments of goat anti-rabbit IgG antibodies, and fluorescein isothiocyanate-conjugated F(ab`) fragments of goat anti-mouse IgG antibodies were obtained from Cappel Laboratories. Mouse monoclonal anti-human TfR antibody (B3/25) (Trowbridge and Omary, 1981) and mouse monoclonal anti-human GT antibody (#8626) (Uemura et al., 1992) were kindly provided by Drs. I. Trowbridge (The Salk Institute, La Jolla, CA) and H. Narimatsu (Soka University, Tokyo, Japan), respectively. Rabbit anti-human GT antiserum was prepared by immunizing a rabbit with soluble form of GT, which was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography column from human milk, according to the established method (Barker et al., 1972).

Transfection and Immunostaining

COS-1 cells were transfected with a cytomegalovirus promoter-driven pcDNAI vectors containing chimeric constructs using DEAE-dextran (Aoki et al., 1992) or DOTAP transfection reagent (Boehringer Mannheim), according to the manufacturer's instruction. For preparation of a stably transfected cell line, CHO cells co-transfected with GT1/2CG and pSVNeo were selected in the presence of 500 µg/ml Geneticin (Life Technologies, Inc.). Resistant clones were isolated and cloned by limiting dilution for selection of cloned cell lines. Expression of the cDNA products was examined by immunofluorescence staining of transfected cells using anti-CG antibodies, as described (Aoki et al., 1992). Merged confocal images were obtained using an MRC-600 Laser-scanning Confocal Imaging system (Bio-Rad).

Immunoprecipitation

COS-1 cells were transfected by plasmid vectors having cDNAs encoding GT, TfR, or chimeric proteins. On day 2 after transfection, cells were first incubated for 1 h in methionine-, cysteine-deficient Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal calf serum, then metabolically labeled for indicated times with [S]methionine and [S]cysteine (100 µCi/ml TranS-label (ICN Radiochemicals), and were chased with complete Dulbecco's modified Eagle's medium. For long term labeling, cells were labeled with 100 µCi/ml TranS-label in the presence of 10 µM methionine and 10 µM cysteine. After 12 h labeling, cells were lysed with lysis buffer (25 mM HEPES, pH 7.4, 1% Nonidet P-40, 10% glycerol, 150 mM NaCl, 0.225 TIU/ml aprotinin, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, 2 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 0.05% NaN). Lysates were preadsorbed with protein G-Sepharose beads (Pharmacia Biotech Inc.) and then reacted with protein G-Sepharose beads precoated with antibodies for 4 h at 4 °C. The beads were washed four times at 4 °C with the washing buffer A (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40, 0.2% sodium deoxycholate, 0.1% SDS), three times at 4 °C with the washing buffer B (10 mM phosphate buffer, pH 7.4, 500 mM NaCl, 0.5% Nonidet P-40), and once at 4 °C with the washing buffer A, and then boiled for 3 min in SDS-PAGE sample buffer. Eluates from the beads were resolved in SDS-PAGE (Laemmli, 1970), and the immunoprecipitates were detected by fluorography.

For digestion with endo--N-acetylglucosaminidase H (endo H), the immunoprecipitates were eluted with 20 mM Tris-HCl buffer, pH 7.5 containing 0.15 M NaCl, 1% SDS, and 10 mM dithiothreitol, diluted with 9 volumes of 0.15 M sodium citrate, pH 5.3, containing 0.5 mM phenylmethylsulfonyl fluoride, and then incubated at 37 °C for 18 h in the presence or absence of 100 milliunits/ml endo H (Boehringer Mannheim). Following trichloroacetic acid precipitation, the proteins were dissolved in SDS sample buffer, electrophoresed, and analyzed by a BAS 2000 radioactive imager (Fuji Film).

Purification and Amino Acid Sequencing of GT-associated Proteins

CHO cells stably transfected with GT1/2CG were cultured in Eagle's medium supplemented with 5% fetal calf serum. Cells collected from 34 culture dishes (15-cm diameter) were solubilized with the lysis buffer described above. After centrifugation, the supernatant was applied to a protein A-Sepharose CL-4B column for preclearing, and subsequently to a rabbit anti-human GT antibodies covalently immunoaffinity (Protein A Sepharose) column, which was prepared as reported (Schneider et al., 1982). After washing with the washing buffers A and B, bound proteins were eluted with 0.1 M glycine, pH 2.5, 0.1% Nonidet P-40. The eluates were immediately neutralized with 1 M Tris, pH 9, and precipitated with 6% trichloroacetic acid. The precipitates were dissolved in SDS-PAGE sample buffer containing 5% -mercaptoethanol, heated to 50 °C for 20 min, and subjected to SDS-PAGE, followed by electrotransfer onto a PVDF membrane (Matsudaira, 1987). After staining with Coomassie Blue, the bands corresponding to 50 and 52 kDa were excised, and the amino-terminal sequences of the proteins were determined on an Applied Biosystems 470A gas-phase sequencer equipped with an on-line phenylthiohydantoin-amino acid analyzer. The analysis was kindly performed by Dr. Kazuo Fujikawa at the University of Washington.

Sucrose Density Gradient Sedimentation

Continuous 5-20% (w/w) sucrose gradients were poured over a 60% (w/w) sucrose cushion, essentially as described (Zagouras et al., 1991), except that all solutions were in 25 mM HEPES, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 0.05% NaN. COS-1 cells expressing chimeric proteins were metabolically labeled and lysed with the lysis buffer as described above. Lysates were loaded on top of the gradients and centrifuged at 38,000 rpm for 19 h at 4 °C. Fractions (0.8 ml) were collected from the top, immunoprecipitated, and electrophoresed followed by fluorography.

RESULTS

Golgi Retention of GT Chimeric Proteins in COS-1 Cells

In our previous studies, the expression of GT chimeras was examined 2 days after transfection by immunofluorescence microscopy, and a double point mutation, Cys Ser and His Leu, within the GT transmembrane domain has been shown to profoundly affect Golgi retention (Aoki et al., 1992). The expression of GT1/2CG protein on cell surface was demonstrated by immuno-staining of intact COS-1 cells followed by fluorescence-activated cell sorter analysis (Aoki et al., 1992). Localization of several different chimeric proteins consisted with the portion of GT (the cytoplasmic tail, the transmembrane domain, and the stem region) and with different reporter molecules (whole CG or the extracellular domain of TfR, see Fig. 1) were tested for their Golgi localization in transfected COS-1 cells by immunofluorescence microscopy. GTCG and GTTfR proteins were mainly localized in the Golgi complex on 2 or 3 days after transfection (see Fig. 2B for GTTfR). Double point mutation (Cys Ser and His Leu) of both these chimeric proteins resulted in diffuse distribution (see Fig. 2, C and D for GTTfR), including cell surface (data not shown; see Aoki et al. (1992)). On the other hand TfR were localized on the cell surface (Fig. 2A), although there was also some staining of the cell interior, including the Golgi complex.


Figure 2: Confocal microscopy of localization of TfR, GTTfR, and GTTfR proteins in COS-1 cells. COS-1 cells were transiently transfected with TfR (A), GTTfR (B), or GTTfR (C and D). On day 2 after transfection, cells were fixed and permeabilized with saponin. TfR proteins and chimeric proteins were stained with monoclonal anti-TfR antibodies followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. The merged pictures of 10-15 Z (tangential) sections of the cells are shown in A (surface expression), B (Golgi localization), and C and D (delocalized expression). In D, the multinucleated cell shows delocalized expression of GTTfR proteins at the left. Simultaneously the cell showing Golgi localization of the proteins is observed at the right of the same field.



Pulse-labeling and chase experiments (Fig. 3) showed that at chase time 0 all of the GTTfR (lane 1) and GTTfR (lane 5) are converted to 75-kDa protein (lanes 2 and 6) by endo H digestion. After 2 h chase, about 40% of GTTfR was converted to 75 kDa band (lane4, lowerband), suggesting that this portion of GTTfR was remained in the ER while the rest of the GTTfR (lane4, upper band) was transported to the Golgi. After 2 h of chase, about 20% of GTTfR mutant was converted to the band of 75 kDa (shown by an arrow in lane8) by endo H, suggesting that this portion of GTTfR was remained in the ER while most of GTTfR (lane8, upperband) was transported to the Golgi. Thus Cys and His residues in the transmembrane domain of GT appear to affect both intracellular transport and Golgi retention.


Figure 3: Endo H digestion of metabolically labeled GTTfR and GTTfR COS-1 cells were transiently transfected with either GTTfR or GTTfR. On day 2 after transfection, cells were pulse-labeled with [S]Met and []Cys for 30 min and chased in regular culture medium for 0 or 2 h as indicated. The chimeric proteins were immunoprecipitated with monoclonal anti-TfR antibodies, and immunoprecipitates were treated with (+) or without (-) endo H prior to analysis by SDS-PAGE. Numbers on the left are molecular markers (kDa).



Dimer Formation Depends on Cysand Hisin the GT Transmembrane Domain

COS-1 cells were transfected with vector alone, plasmid vectors encoding TfR, GTTfR, or GTTfR (Fig. 1). On day 2 after transfection, COS-1 cells were labeled with [S]Met and [S]Cys for 1 h, followed by a 2-h chase period, and were immunoprecipitated using anti-TfR antibodies. When the immunoprecipitates were analyzed by SDS-PAGE, all mature proteins of TfR, GTTfR, and GTTfR were detected as approximately 90-kDa bands under reducing conditions (Fig. 4). The formation of dimers (approximately 180 kDa, shown by an arrowhead in lane 3) was observed in the cells transfected with GTTfR (lane 3) but not with GTTfR (lane 2). These results suggest that GTTfR polypeptides are forming homodimers in these cells, which appear to be relatively resistant to SDS and -mercaptoethanol. These results also show that the dimer formation is largely dependent on the presence of Cys and His residues in the transmembrane domain of GT.


Figure 4: Fluorogram of SDS-PAGE of immunoprecipitate. COS-1 cells were transiently transfected with either vector alone (lane1), TfR (lane2), GTTfR (lane3), or GTTfR (lane4). On day 2 after transfection, cells were pulse-labeled with [S]Met and [S]Cys for 1 h followed by 2 h of chase. Cell lysates were immunoprecipitated with monoclonal anti-TfR antibodies in the presence of protein G-Sepharose beads. The beads were washed as described under ``Materials and Methods.'' The immunoprecipitates were boiled in SDS-PAGE sample buffer in the presence of 5% -mercaptoethanol, analyzed by SDS-PAGE on a 6.5% gel, and detected by fluorography. Molecular markers are shown in kDa.



Because Cys and His residues are often involved in metal chelating as exemplified in zinc finger proteins (Beng, 1990), the effect of EDTA and o-phenanthroline on the dimers were examined. Thus the immunoprecipitates of GTTfR were boiled with 50 mM EDTA or 10 mMo-phenanthroline in the presence of SDS and -mercaptoethanol just prior to SDS-PAGE. EDTA and o-phenanthroline, however, did not dissociate the dimers to monomers (data not shown). These observations suggest that metal chelating is not involved in the dimer formation between GT molecules. Dimer formation of GT is probably due to the secondary structure induced by a unique conformation of peptide within the lipid bilayer (Bormann et al., 1989; Machamer, 1993).

Association of GT with Cellular Proteins

In parallel to the above described experiments, we examined whether any proteins are associated with GT. We performed extensive co-immunoprecipitation analysis using GT and three GT chimeric proteins, GT1/2CG, GTCG, and GTTfR (see Fig. 1 for chimeras). COS-1 cells were transfected with plasmid vectors containing these cDNAs. On day 2 after transfection, COS-1 cells were labeled with [S]Met and [S]Cys, and cell lysate was subjected for immunoprecipitation.

When the immunoprecipitates using anti-TfR antibodies were analyzed by SDS-PAGE (Fig. 5, lanes 1-3), monomer and dimer of GTTfR were detected as 90-kDa (lane 3, asterisk) and 180-kDa (lane 3, arrowhead) bands, respectively. TfR, which can pass through the Golgi complex without apparent Golgi retention, was used as a control. COS-1 cells transfected with GT were also included as a control to detect any nonspecific component co-immunoprecipitated with anti-TfR antibodies. In these analyses, we repeatedly found that two proteins at approximately 50 and 52 kDa (Fig. 5, arrows) were co-immunoprecipitated with the portion of GT molecule that contains the cytoplasmic tail, transmembrane domain, and stem region. The 50- and 52-kDa proteins were not detected or were only faintly detectable in the immunoprecipitates of TfR (lane2) and that of control (lane1).


Figure 5: Co-immunoprecipitation of GT with cellular proteins. COS-1 cells were transiently transfected with GT (lane1), TfR (lane2), GTTfR (lane3), GT1/2CG (lane4), GT (lane5), and TfR (lane6). On day 2 after transfection, cells were labeled with [S]Met and [S]Cys for 21 h. Cell lysates were immunoprecipitated with monoclonal anti-TfR antibodies (lanes1-3) or monoclonal anti-GT antibodies (lanes 4-6) in the presence of protein G-Sepharose beads. The immunoprecipitates were analyzed as described in Fig. 4. The positions of monomers and dimers of each chimeric protein are shown by asterisks and arrowheads, respectively. Arrows indicate the major co-immunoprecipitated materials with the proteins possessing the portion of GT structure (see Fig. 1). Molecular markers are shown in kDa.



COS-1 cells transfected with GT1/2CG, GT, and TfR were subjected for immunoprecipitation using polyclonal anti-GT antibodies (Fig. 5, lanes 4-6). GT1/2CG proteins were detected at approximately 66, 63, and 57 kDa (monomers; asterisks in lane4) and at 132, 126, and 114 kDa (dimers; arrowheadsin lane4). The difference in these molecular sizes are most likely caused by diffferences in glycosylation. GT was detected at 52 kDa (monomer; asterisk in lane5) and 104 kDa (dimer; arrowhead in lane5). The 50- and 52-kDa proteins were detected again in the immunoprecipitate of GT1/2CG (lane4). Although these two co-immunoprecipitated proteins are not clearly visible in the immunoprecipitates of GT (Fig. 5, lane5), this is because 50- and 52-kDa proteins overlap with GT proteins on SDS-PAGE. The 50- and 52-kDa proteins were not detected or only faintly detectable in the control (lane6).

These results (Fig. 5) indicate that, regardless of the antibodies or reporter molecules used, co-precipitation of 50- and 52-kDa proteins depends on the portion of GT molecule consisted with cytoplasmic tail, transmembrane domain, and stem region.

Identification of the 50- and 52-kDa Proteins

To identify the 50- and 52-kDa proteins that co-immunoprecipitated with GT, these proteins were purified and their amino acid sequences were determined. The 50-kDa and 52-kDa proteins were co-purified with GT from the lysate of CHO cells stably expressing GT1/2CG by affinity chromatography using an anti-GT antibody column (see details under ``Materials and Methods''). The immunopurified materials were separated by SDS-PAGE, electrotransferred onto a PVDF membrane, and subsequently subjected for amino-terminal sequence analysis. Fig. 6shows the Coomassie Blue staining of the gel and the amino-terminal sequences determined. The 52- and 50-kDa proteins were found identical to - and -tubulin, respectively. In addition, amino acid sequence confirmed the 57-kDa protein as GT.


Figure 6: Purification of GT1/2CG and 50 kDa and 52 kDa proteins. CHO cells stably transfected with GT1/2CG were solubilized. Cell lysates were applied onto a protein A-Sepharose column for preclearing. Bound materials were eluted and analyzed by SDS-PAGE. Lane 1 shows a Coomassie Blue staining pattern as a control. Then unbound materials from a protein A-Sepharose column were applied onto a rabbit anti-GT antibody-conjugated protein A-Sepharose column. After washing, bound materials were eluted and analyzed by SDS-PAGE. Lane2 shows the gel stained by Coomassie Blue. Subsequently, the immunoaffinity-purified materials were subjected to SDS-PAGE and electrotransferred onto a PVDF membrane. After staining with Coomassie Blue, the corresponding bands were excised and the amino-terminal sequences of the proteins were determined. The determined sequences are illustrated at the right. X indicates no signal, and parentheses indicate the most likely amino acid residues. Molecular markers are indicated in kDa.



Association of - and -Tubulins with GT Dimers and Oligomers

Since there were reports describing that the oligomerization is an important factor for Golgi retention of the resident proteins (Nilsson et al., 1994; Swift and Machamer, 1991), we examined the relationship between oligomerization of GT and association of tubulins.

COS-1 cells were transfected with GTTfR, labeled with [S]Met and [S]Cys, and subsequently solubilized. Lysates were loaded onto 5-20% sucrose density gradients, centrifuged, and fractionated into 13 fractions. The sucrose gradient centrifugation was calibrated using molecular size markers, thus the fractions which should contain GT monomer and dimer were determined to be fractions 2/3 and 4/5, respectively. S-Labeled proteins in each fraction were subjected to immunoprecipitation using anti-TfR antibodies, and immunoprecipitates were analyzed by SDS-PAGE. As shown in Fig. 7, monomers of GTTfR (approximately 90 kDa) were detected in fraction 2 and later fractions. Dimers of GTTfR (approximately 180 kDa) were detected in fraction 4 and later fractions. The GT polypeptides fractionated in tubes later than fraction 5 are considered as oligomers larger than trimers. Therefore the result shown in Fig. 7indicates that a substantial quantity of GTTfR polypeptides in the cell lysate behaved as if they are large oligomers. The result (Fig. 6) also shows that - and -tubulins appear only in fraction 7 and later fractions, suggesting that - and -tubulins associate only with large oligomers of GT but not with a monomer.


Figure 7: Sucrose density gradient sedimentation of GTTfR. COS-1 cells were transiently transfected with GTTfR. On day 2 after transfection, cells were pulse-labeled with [S]Met and [S]Cys for 1 h followed by 2 h of chase. Cell lysates were subjected to sucrose-density gradient centrifugation. The gradient was split into 13 equal volume fractions. GTTfR was immunoprecipited and analyzed by SDS-PAGE followed by fluorography. Twolongarrows indicate - and -tubulins. A shortarrow and arrowhead indicate monomer and dimer of GT. Fractions that are expected to contain GT monomer, dimer, and oligomers (larger than trimer) are indicated as mono, di, and oligo, respectively. Molecular markers are shown in kDa.



DISCUSSION

Recent studies indicate that the transmembrane domain and adjacent cytoplasmic and luminal domains of Golgi resident proteins are largely responsible for these proteins to be retained in the Golgi complex (Machamer and Rose, 1987; Munro, 1991; Nilsson et al., 1991; Swift and Machamer, 1991; Aoki et al., 1992; Burke et al., 1992; Colley et al., 1992; Russo et al., 1992; Tang et al., 1992; Teasdale et al., 1992; Wong et al., 1992). However, it is not yet clear how these transmembrane and adjacent domains lead to Golgi retention.

In this paper, we investigate the molecular mechanism of Golgi retention by using GT as a model system. We found that the double point mutation (Cys Ser and His Leu) within the transmembrane domain of GT, reduced Golgi retention of the molecule significantly (Fig. 2; see also Aoki et al. (1992)). As shown in Fig. 4, the presence of these two amino acid residues is critical for dimer formation. The formation of homodimers may facilitate further oligomerization of GT (Fig. 7), which could result in apparent immobilization of GT in the Golgi membrane.

Our co-immunoprecipitation experiments demonstrated that GT oligomers associate with - and -tubulins (Fig. 7). Microtubules, composed of filamentous polymeric assemblies of a heterodimer of one - and one -tubulin polypeptides, are cytoskeletal components of cells and play a central role in rapid organelle movements, which occur in such processes as vesicle fusion and transport in some vesicle fusion events, and in some steps of transport (Allen et al., 1982; Lippincott-Schwartz et al., 1990). In addition, microtubules are involved in maintaining the Golgi structure since disruption of the microtubule network during mitosis or drug-induced disassembly leads to fragmentation and scattering of Golgi complex throughout the cell (Turner and Tartakoff, 1989; Kreis, 1990; Iida and Shibata, 1991). Reassembly of the microtubule network results in reaggregation of the Golgi complex at the microtubule-organizing center (Ho et al., 1989). Microtubules are the only cytoskeletal elements for which structural and some functional relationships to the Golgi complex have been established. Accordingly, it is possible that - and -tubulins actively participate in the organization of the Golgi complex, thus playing a role in retention of resident proteins in the Golgi complex.

Presently, we do not know how GTs interact with - and -tubulins. We envisage that GTs form homodimers (Fig. 4, 5, and 7), which subsequently further aggregate to form a large complex in the Golgi membrane (Fig. 7). Such a complex may associate with proteins present in the cytoplasm, in the membrane, and in the lumen of the Golgi. As shown in this study, the dimer formation and subsequent oligomerization may be mediated primarily by the transmembrane domain. The GT oligomers may then anchor either directly or indirectly to the bundle of tubulins. As tubulins are a part of microtubule-based motor proteins, Golgi retention and transport of GT could be supported and guided by a microtubule-based network as has been suggested previously (Strous et al., 1991). Because tubulins localize in the cytoplasm, the association of GT with tubulins may be mediated by the cytoplasmic tail of GT. Deletion of the cytoplasmic tail caused leakage of GT to the plasma membranes (Nilsson et al., 1991; Aoki et al., 1992), supporting the hypothesis that Golgi retention of GT is stabilized by its association with cytoplasmic component. However, association of tubulin may not be the cause of Golgi retention but rather the result of it, because GT and tubulin are co-localized in HeLa cells, which were treated with brefeldin A (Strous et al., 1991). Furthermore, our preliminary data showed that GT mutant proteins were also associated with - and -tubulins, although careful analysis should be carried out to define differences, if any are present, between GT proteins at the Golgi and at cell surface. It is possible that other proteins are involved in the association between GT and tubulins. In fact, we have detected five additional proteins co-immunoprecipitated with GT and GT chimeras but not with TfR.() It is not presently clear whether or not other Golgi enzymes also associate with tubulins. Slusarewicz et al.(1994) identified a functional medial Golgi ``matrix,'' which could promote binding of medial Golgi enzymes. The matrix includes several proteins, although they appear not to include - and - tubulins, and GT did not bind to the matrix. It is therefore possible that the association of tubulins is unique to GT or trans-Golgi enzymes.

Currently, two models have been proposed for the mechanism of Golgi retention, which are not mutually exclusive. (i) Golgi enzymes form large hetero- and/or homo-oligomers via their lumenal and transmembrane domains by ``kin recognition.'' The oligomers attach to the matrix via the cytoplasmic domains, leading to prevent Golgi enzymes from entering into the budding transportation vesicles (Weisz et al., 1993; Nilsson et al., 1994; Slusarewicz et al., 1994). (ii) Retention depends on the length of transmembrane domains and the lipid composition of the membranes in the Golgi complex; increasing the length of the transmembrane domain could direct Golgi enzymes to the plasma membrane (Munro, 1991; Masibay et al., 1993; Bretscher and Munro, 1993; Pelham and Munro, 1993).

The results obtained by present study are consistent with the first model, in which oligomerization of proteins is an important factor for being retained in the Golgi complex. The present study also suggests that GT oligomers are stabilized in the Golgi by their direct or indirect association with tubulins and that the Golgi retention is a part of the intracellular process controlled by the cytoskeleton.


FOOTNOTES

*
This work was supported in part by Grant R01-DK37016 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a research fellowship from the Uehara Memorial Foundation (Tokyo, Japan). Present address: Dept. of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860, Japan.

To whom correspondence should be addressed: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.

The abbreviations used are: ER, endoplasmic reticulum; GT, UDP-galactose:-D-N-acetylglucosaminide -1,4-galactosyltransferase; TfR, transferrin receptor; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; endo H, endo--N-acetylglucosaminidase H; PVDF, polyvinylidene difluoride.

N. Yamaguchi and M. N. Fukuda, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Ian S. Trowbridge, John K. Rose, and Hisashi Narimatsu for their gifts of antibodies and cDNAs. We also thank Dr. Kazuo Fujikawa (University of Washington, Seatle, WA) for amino acid sequencing; Drs. Harry Schachter (The Hospital for Sick Children, Toronto, Canada), Minoru Fukuda, and Yu Yamaguchi (La Jolla Cancer Research Foundation) for their critical reading of the manuscript; and Scott Armstrong for technical assistance.


REFERENCES
  1. Allen, R. D., Metuzals, J., Tasaki, I., Brady, S. T., and Gilbert, S. P.(1982) Science 218, 1127-1128 [Medline] [Order article via Infotrieve]
  2. Aoki, D., Appert, H. E., Johnson, D., Wong, S. S., and Fukuda, M. N. (1990) EMBO J. 9, 3171-3178 [Abstract]
  3. Aoki, D., Lee, N., Yamaguchi, N., Dubois, C., and Fukuda, M. N.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4319-4323 [Abstract/Free Full Text]
  4. Barker, R., Olsen, K. W., Shaper, J. H., and Hill, R. L.(1972) J. Biol. Chem. 247, 7135-7147 [Abstract/Free Full Text]
  5. Bayna, E. M., Shaper, J. H., and Shur, B. D.(1988) Cell 53, 145-157 [Medline] [Order article via Infotrieve]
  6. Beng, J. M.(1990) J. Biol. Chem. 265, 6513-6516 [Free Full Text]
  7. Beyer, T. A., and Hill, R. L.(1982) in The Glycoconjugates (Horowitz, M., ed) Vol. III, Part A, pp. 25-45, Academic Press, New York
  8. Bormann, B.-J., Knowles, W. J., and Marchesi, V. T.(1989) J. Biol. Chem. 264, 4033-4037 [Abstract/Free Full Text]
  9. Bretscher, M. S., and Munro, S.(1993) Science 261, 1280-1281 [Medline] [Order article via Infotrieve]
  10. Burke, J., Pettitt, J. M., Schachter, H., Sarkar, M., and Gleeson, P. A.(1992) J. Biol. Chem. 267, 24433-24440 [Abstract/Free Full Text]
  11. Colley, K. J., Lee, E. U., and Paulson, J. C.(1992) J. Biol. Chem. 267, 7784-7793 [Abstract/Free Full Text]
  12. D'Agostaro, G., Bendiak, B., and Tropak, M.(1989) Eur. J. Biochem. 183, 211-217 [Abstract]
  13. Eckstein, D. J., and Shur, B. D.(1992) Exp. Cell. Res. 201, 83-90 [Medline] [Order article via Infotrieve]
  14. Fiddes, J. C., and Goodman, H. M.(1979) Nature 281, 351-356 [Medline] [Order article via Infotrieve]
  15. Ho, W. C., Allan, V. J., van Meer, G., Berger, E. G., and Kreis, T. (1989) Eur. J. Cell Biol. 48, 250-263 [Medline] [Order article via Infotrieve]
  16. Iida, H., and Shibata, Y.(1991) J. Histochem. Cytochem. 39, 1349-1355 [Abstract]
  17. Kreis, T.(1990) Cell Motil. Cytoskel. 15, 67-70 [Medline] [Order article via Infotrieve]
  18. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  19. Lewis, M. J., and Pelham, H. R. B.(1992) Cell 68, 353-364 [Medline] [Order article via Infotrieve]
  20. Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H.-P., Yuan, L. C., and Klausner, R. D.(1990) Cell 60, 821-836 [Medline] [Order article via Infotrieve]
  21. Lopez, L. C., Bayna, E. M., Litoff, D., Shaper, N. L., Shaper, J. H., and Shur, B. D.(1985) J. Cell Biol. 101, 1501-1510 [Abstract]
  22. Machamer, C. E.(1993) Curr. Opin. Cell Biol. 5, 606-612 [Medline] [Order article via Infotrieve]
  23. Machamer, C. E., and Rose, J. K.(1987) J. Cell Biol. 105, 1205-1214 [Abstract]
  24. Masibay, A. S., Balaji, P. V., Boeggeman, E. E., and Qasba, P. K. (1993) J. Biol. Chem. 268, 9908-9916 [Abstract/Free Full Text]
  25. Masri, K. A., Appert, H. E., and Fukuda, M. N.(1988) Biochem. Biophys. Res. Commun. 157, 657-663 [Medline] [Order article via Infotrieve]
  26. Matsudaira, P.(1987) J. Biol. Chem. 262, 10035-10038 [Abstract/Free Full Text]
  27. Munro, S.(1991) EMBO J. 10, 3577-3588 [Abstract]
  28. Nakazawa, K., Ando, T., Kimura, T., and Narimatsu, H.(1988) J. Biochem. (Tokyo) 104, 165-168 [Abstract]
  29. Nilsson, T., Lucocq, J. M., Mackay, D., and Warren, G.(1991) EMBO J. 10, 3567-3575 [Abstract]
  30. Nilsson, T., Hoe, M. H., Slusarewicz, P., Rabouille, C., Watson, R., Hunte, F., Watzele, G., Berger, E. G., and Warren, G.(1994) EMBO J. 13, 562-574 [Abstract]
  31. Palade, G. E.(1975) Science 189, 347-358 [Medline] [Order article via Infotrieve]
  32. Paulson, J. C., and Colley, K. J.(1989) J. Biol. Chem. 264, 17615-17618 [Free Full Text]
  33. Pelham, H. R. B., and Munro, S.(1993) Cell 75, 603-605 [Medline] [Order article via Infotrieve]
  34. Pfeffer, S. R., and Rothman, J. E.(1987) Annu. Rev. Biochem. 56, 829-852 [CrossRef][Medline] [Order article via Infotrieve]
  35. Roth, J., and Berger, E. G.(1982) J. Cell Biol. 92, 223-229
  36. Roth, J., Lentze, M. J., and Berger, E. G.(1985) J. Cell Biol. 100, 118-125 [Abstract]
  37. Rothman, J. E., and Orci, L.(1992) Nature 355, 409-415 [CrossRef][Medline] [Order article via Infotrieve]
  38. Russo, R. N., Shaper, N. L., Taatjes, D. J., and Shaper, J. H.(1992) J. Biol. Chem. 267, 9241-9247 [Abstract/Free Full Text]
  39. Schachter, H., and Roseman, S.(1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 85-160, Plenum Press, New York
  40. Schneider, C., Newman, R. A., Sutherland, D. R., Asser, U., and Greaves, M. F.(1982) J. Biol. Chem. 257, 10766-10769 [Abstract]
  41. Schneider, C., Owen, M. J., Banville, D., and Williams, J. G.(1984) Nature 311, 675-678 [Medline] [Order article via Infotrieve]
  42. Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H.(1988) J. Biol. Chem. 263, 10420-10428 [Abstract/Free Full Text]
  43. Slusarewicz, P., Nilsson, T., Hui, N., Watson, R., and Warren, G. (1994) J. Cell Biol. 124, 405-413 [Abstract]
  44. Strous, G. J.(1986) CRC Crit. Rev. Biochem. 21, 119-151 [Medline] [Order article via Infotrieve]
  45. Strous, G. J., and Berger, E. G.(1982) J. Biol. Chem. 257, 7623-7628 [Free Full Text]
  46. Strous, G. J., Berger, E. G., van Kerkhof, P., Bosshart, H., Berger, B., and Geuze, H. J.(1991) Biol. Cell 71, 25-31 [Medline] [Order article via Infotrieve]
  47. Suganuma, T., Muramatsu, H., Muramatsu, T., Ihida, K., Kawano, J., and Murata, F.(1991) J. Histochem. Cytochem. 39, 299-309 [Abstract]
  48. Swift, A. M., and Machamer, C. E.(1991) J. Cell Biol. 115, 19-30 [Abstract]
  49. Tang, B. L., Wong, S. H., Low, S. H., and Hong, W.(1992) J. Biol. Chem. 267, 10122-10126 [Abstract/Free Full Text]
  50. Teasdale, R. D., D'Agostaro, G., and Gleeson, P. A.(1992) J. Biol. Chem. 267, 4084-4096 [Abstract/Free Full Text]
  51. Trowbridge, I. S., and Omary, M. B.(1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3039-3043 [Abstract]
  52. Turner, J. R., and Tartakoff, A. M.(1989) J. Cell Biol. 109, 2081-2088 [Abstract]
  53. Uemura, M., Sakaguchi, T., Uejima, T., Nozawa, S., and Narimatsu, H. (1992) Cancer Res. 52, 6153-6157 [Abstract]
  54. Walter, P., and Lingappa, V. R.(1986) Annu. Rev. Biochem. 2, 499-516
  55. Weisz, O. A., Swift, A. M., and Machamer, C. E.(1993) J. Cell Biol. 122, 1185-1196 [Abstract]
  56. Wong, S. H., Low, S. H., and Hong, W.(1992) J. Cell Biol. 117, 245-258 [Abstract]
  57. Zagouras, P., Ruusala, A., and Rose, J. K.(1991) J. Virol. 65, 1976-1984 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.