Journal of Science 108, 1617-1627 (1995) 1617 Printed in Great Britain © The Company of Biologists Limited 1995

Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides

Catherine Rabouille, Norman Hui, Felicia Hunte, Regina Kieckbusch, Eric G. Berger*, Graham Warren and Tommy Nilsson† Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK *Present address: Physiologisches Institut, Universität Zürich, Switzerland †Author for correspondence

SUMMARY

The distribution of β1,2 N-acetylglucosaminyltransferase I tion but they occupied the trans cisterna and the trans- (NAGT I), α1,3-1,6 mannosidase II (Mann II), β1,4 galac- Golgi network (TGN). These results generalise our earlier tosyltransferase (GalT), α2,6 sialyltransferase (SialylT) observations on the overlapping distribution of Golgi was determined by immuno-labelling of cryo-sections from enzymes and show that each of the trans compartments of HeLa cell lines. Antibody labelling in the HeLa cell line was the in HeLa cells contains unique mixtures made possible by stable expression of epitope-tagged forms of those Golgi enzymes involved in the construction of of these or forms from species to which specific complex, N-linked oligosaccharides. antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae Key words: Golgi apparatus, trans-Golgi network, of the stack. GalT and SialylT also had the same distribu- glycosyltransferase, mannosidase, TGN38

INTRODUCTION two adjacent cisternae was taken as evidence of cisternal dupli- cation. This interpretation was supported by the observation The construction of complex, bi-antennary, N-linked oligosac- that the number of cisternae in the Golgi stack can vary widely charides involves the sequential action of enzymes located in from tissue to tissue and from organism to organism (see different parts of the Golgi apparatus (for reviews, see Kornfeld Fawcett, 1981). To investigate the possibility of cisternal and Kornfeld, 1985; Roth, 1991). α1,2 mannosidase I continues duplication, we examined, using immunogold electron the trimming of mannose residues that started in the endoplas- microscopy, the distribution of NAGT I and GalT in HeLa mic reticulum (ER) leaving a penta-mannose core to which the cells. Each enzyme was found in two, adjacent cisternae but, first N-acetylglucosamine is added by β1,2 N-acetylglu- contrary to expectation, both were present in the trans cisterna cosaminyltransferase I (NAGT I). α1,3-1,6 mannosidase II (Nilsson et al., 1993). In other words, the two enzymes had an (Mann II) removes two more mannose residues permitting overlapping distribution such that each cisterna contained a addition of the final N-acetylglucosamine by β1,2 N-acetylglu- unique mixture of enzymes not a unique set. In order to gen- cosaminyltransferase II. Each branch can then be elongated by eralise these observations and obtain further evidence against the addition of galactose by β1,4 galactosyltransferase (GalT) cisternal duplication and in favour of each cisterna having a and sialic acid by α2,6 sialyltransferase (SialylT). Fucose may unique composition, we have extended our observations to also be added prior to or following the addition of sialic acid. other Golgi enzymes. To do this we have generated a series of GalT was the first of these enzymes to be localised, first to stable HeLa cell lines expressing either epitope-tagged the trans cisterna (Roth and Berger, 1982) and later to the enzymes or ones to which antibodies were available. trans-Golgi network (TGN) (Lucocq et al., 1989; Nilsson et al., 1993). SialylT was found to localise to the trans Golgi cisterna and the TGN (Roth et al., 1985) and to have the same MATERIALS AND METHODS distribution in most though not all cells (Roth et al., 1986). Stable cell lines NAGT I was found in medial cisternae (Dunphy et al., 1985) as, more recently, was Mann II (Velasco et al., 1993). The HeLa cell lines expressing either human NAGT I (Kumar et al., 1990) tagged with a myc-epitope or murine Mann II (Moremen and Robbins, location of these enzymes strongly supported the idea that 1991) have been described elsewhere (Nilsson et al., 1993, 1994). proteins undergoing transport moved through the stack in a Human SialylT (Grundmann et al., 1990) was tagged with a VSV- cis to trans direction, sampling each of the compartments in G epitope (underlined) (Kreis, 1986; Soldati and Perriard, 1991) using turn. PCR (Saiki et al., 1988) to introduce the epitope immediately prior to The fact that most of these enzymes were usually found in the stop codon. Primers used were:

1618 C. Rabouille and others

Table 1. Stable HeLa cell lines used in this study Antibody used to detect the Expression level relative Cell line Origin of expressed Epitope tag stably-expressed protein(s) to endogenous protein* NAGT I-HeLa Human myc 9E.10 monoclonal 4-fold Mann II-HeLa Mouse None Polyclonal anti-murine Mann II 6-fold SialylT-HeLa Human VSV-G P5D4 monoclonal 50 to 100-fold TGN38/SialylT-HeLa SialylT Human VSV-G P5D4 monoclonal 10-fold TGN38 Rat None Polyclonal anti-rat TGN38 2.5-fold

*Measured by the increase in enzyme activity (NAGT I and SialylT) or by comparing the linear density of gold labelling over the Golgi with that in NRK cells (Mann II and TGN38). Parental HeLa cells were transfected with pSRα containing the appropriate cDNA and stable lines selected as described in Materials and Methods.

5′GTCGACGGATCCACCATGATTCACACCAACCTGAAG3′; Laboratories, Cardiff, UK) were used to detect the primary mono- and clonal antibodies whereas goat anti-rabbit antibodies coupled to gold 5′GTCGACGGATCCTTACTTTCCCAGCCTGTTCATCTCTA- (Biocell Research Laboratories, Cardiff, UK) or Protein A gold (from TATCGGTGTAAGGGCAGTGAATGGTCCGGAAGCC3′. Dept of Cell Biology, Utrecht School of Medicine, Utrecht, the The PCR product was sequenced and subcloned into the BamHI Netherlands) were used to detect primary polyclonal antibodies. site of pSRα (DNAX, Palo Alto, CA). Two protocols were used for double-labelling experiments. When A full length cDNA encoding rat TGN38 (Luzio et al., 1990) was one of the primary antibodies was a monoclonal and the other a poly- digested with HindIII and complementary oligonucleotides encoding clonal, they were mixed together for the initial incubation with the a BamHI site were introduced immediately following the stop codon. section. Each of the secondary antibodies was then added sequentially The complementary oligonucleotides were: (Nilsson et al., 1993). When both antibodies were polyclonal, incu- 5′AGCTTTGAG3′ and 5′GATCCTCAA3′. bation with the first primary antibody was followed by goat anti-rabbit The coding region of TGN38 was then excised and subcloned into or Protein A coupled to one size of gold. Sections were then fixed for the BamHI site of pCMUIV (Nilsson et al., 1989). 15 minutes in 4% paraformaldehyde and the second primary antibody HeLa cells were transfected with tagged SialylT in pSRα either added followed by goat anti-rabbit or Protein A coupled to a different alone or together with TGN38 in pCMUIV. Transfection and isolation size of gold (Slot et al., 1991). of stable lines was carried out essentially as described previously Grids were stained with 2% neutral uranyl acetate and embedded (Nilsson et al., 1994). Table 1 summarises relevant properties of the in 2% methyl cellulose containing 0.4% uranyl acetate as described HeLa cell lines used. Membrane fractionation and western blotting SialylT-HeLa and TGN38/SialylT-HeLa cells were grown in NAGT I N C Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supple- 6 23 418 mented with 10% foetal calf serum, penicillin (100 µg/ml), strepto- mycin (100 µg/ml) and geneticin (500 µg/ml) (Gibco). Approximately 109 cells were used to isolate Golgi membranes (Balch et al., 1984) which were purified at least 10-fold over homogenate assayed by MannII NC GalT activity (Bretz and Stäubli, 1977). Protein concentration was determined using the BCA protein assay kit (Pierce Chemical Co, 5 21 1124 Rockford, IL). SDS-PAGE was carried out essentially as described by Blobel and Dobberstein (1975) and western blotting as described previously (Nilsson et al., 1993). SialylT was assayed as described by Dunphy et al. (1981) using asialo-transferrin as the substrate. GalT NC Immunogold electron microscopy 24 20 354 Cells were fixed either in 2% paraformaldehyde and 0.2% glu- taraldehyde in 0.1 M phosphate buffer, pH 7.4, or in 0.5% glu- taraldehyde in the same buffer containing 0.2 M sucrose and processed as described previously (Rabouille et al., 1993). Briefly, SialylT N C cells were embedded in 10% gelatine and small blocks were infused 9 17 380 with 2.3 M sucrose and frozen in liquid nitrogen. Ultra-thin cryosec- tions were cut on an Ultracut E microtome with FC4E cryo-attach- ment and transferred onto collodion-carbon coated, copper or nickel, 100-mesh grids. All antibodies and gold conjugates were diluted in TGN38 C N 0.5% fish skin gelatine in PBS. 34 19 287 The following primary antibodies were used: the 9E.10 mouse monoclonal antibody which recognises the c-myc epitope (Evan et al., Fig. 1. Topology of the hybrid proteins stably expressed in HeLa 1985) at the C-terminus of NAGT I; a rabbit polyclonal antibody cells. Parental HeLa cells were selected for stable expression of recognising rat Mann II (Moremen et al., 1991); a rabbit polyclonal NAGT I, Mann II, SialylT or TGN 38 (together with SialylT). The antibody (N11) recognising human GalT (Watzele et al., 1991); the topology of the endogenous GalT is presented for comparison. The P5D4 mouse monoclonal antibody which recognises the VSV-G tag numbers (from left to right) refer to the length (not to scale) of the (Kreis, 1986; Soldati and Perriard, 1991) at the C-terminus of SialylT; cytoplasmic tail, the membrane-spanning domain, the lumenal and a rabbit polyclonal antibody recognising rat TGN38 (Luzio et al., domain and the epitope tag (where present). Note that all the Golgi 1990). Goat anti-mouse antibodies coupled to gold (Biocell Research enzymes are type II proteins whereas TGN38 is type I. Mapping the distribution of resident Golgi proteins 1619

kDa

54 58 kDa kDa

Fig. 2. Western blotting of the SialylT-HeLa and TGN38/SialylT- HeLa cell lines. Golgi membranes were isolated from each cell line, fractionated by SDS-PAGE, blotted and probed for TGN38 (lane3) and/or SialylT (lanes 1 and 2). Equal amounts were loaded in each lane. by Tokuyasu (1980). Grids were examined at 80 kV using a Philips CM10 electron microscope. Pictures were taken at a magnification of 15.5 or 21 K. Quantitation Definitions The compartments of the Golgi apparatus were defined as described previously (Nilsson et al., 1993; Ponnambalam et al., 1994). Briefly, the trans or T cisterna is defined as the last continuous cisterna on the Fig. 3. Immunofluorescence microscopy of the SialylT-HeLa and side of the Golgi stack that labels for GalT. Since the Golgi stack in TGN38/SialylT-HeLa cell lines. (A) SialylT-HeLa cells were fixed, HeLa cells typically contains three cisterna, the T-1 cisterna most likely permeabilised and labelled for SialylT. Note specific labelling of a corresponds to the medial cisterna and the T-2 to the cis cisterna. The compact, juxta-nuclear reticulum. (B) TGN38/SialylT-HeLa cells T+1 compartment is the TGN and comprises a tubulo-reticular network were fixed, permeabilised and labelled for TGN38. Note the punctate closely apposed to the trans face of the trans cisterna. It differs from labelling in addition to labelling of a compact, juxta-nuclear the CGN (T-3) in having clathrin-coated (Pearse and Robinson, 1990) reticulum. Bar, 10 µm. in addition to COP-coated buds. Clathrin coats have a different mor- phology and thickness to COP coats (Orci et al., 1984, 1985; Oprins et al., 1993). Nevertheless, it was occasionally difficult to distinguish the the surface density was estimated by the point-hit method (Weibel, TGN from the CGN so double-labelling for GalT and the test enzyme 1979; Ponnambalam et al., 1994). The length of every portion of was used in preliminary experiments to define the polarity of the stack. membrane within this boundary was estimated by the intersection method (Weibel, 1979; Nilsson et al., 1993). Since the ratio of surface Relative distribution density to length was found to be constant (0.062±0.02) between The relative distribution of gold particles over the TGN and each different cell lines, the membrane length in most experiments was cal- cisterna was estimated by counting the number of gold particles culated from the surface density and this ratio. falling within the boundary of each structure. The boundary of a The grid had a 5 mm spacing and the micrograph a final magnifi- cisterna was defined as the cisternal membrane. The boundary of the cation that varied from 50 to 100 K. The linear density was calculated TGN was defined as the interface between the outermost membranes by dividing the number of gold particles by the membrane length. of the tubulo-reticular network and the immediately adjacent amorphous and was drawn on each micrograph. On Indirect immunofluorescence occasion this boundary would include profiles of budded vesicles TGN38/SialylT-HeLa cells were grown to 70% confluency on cover which were included in the quantitation whilst other structures (e.g. slips and incubated for 2 hours in the presence of 10 µg/ml cyclo- vacuolar ) were omitted. heximide. Cells were fixed and permeabilised essentially as described by Louvard et al. (1982). Bound primary antibodies were visualised Linear density using secondary antibodies coupled either to Texas Red (Vector Lab- The linear density of gold particles/µm membrane was estimated as oratories, Inc., Burlingame, CA) or FITC (Dakopatts, Copenhagen). described previously for the T, T-1 and T-2 cisternae (Nilsson et al., Cells were visualised using a Zeiss Axiophot Epifluorescence micro- 1993). The boundary of the TGN was drawn on each micrograph and scope and photographed directly using Ilford black and white film. 1620 C. Rabouille and others

RESULTS The expression levels relative to endogenous protein were estimated in one of two ways and are summarised in Table 1. Characterisation of the stable cell lines For NAGT I and SialylT the activity of the enzyme was GalT was the only endogenous enzyme under study that could measured and compared to the expression level of the endoge- be readily detected in cryo-sections of HeLa cells using nous protein in the parental HeLa cell line. Mann II activity affinity-purified antibodies to the deglycosylated protein could not be estimated in the same way because there were too (Watzele et al., 1991). Detection of the other enzymes was many contaminating activities in whole cell homogenates. The made possible by transfecting the parental HeLa cell line with level was, therefore, estimated by immuno-gold microscopy the PSRα plasmid containing the appropriate cDNA (see and compared with the level of the endogenous protein in NRK Materials and Methods) and selecting stable cell lines in the cells. The same procedure was used for TGN38 which has no presence of geneticin. Clones were picked at random and known activity that could be measured. immunofluorescence microscopy was used to select those The level of over-expression varied widely (Table 1). At the clones expressing approximately equal amounts of protein in lower end was TGN38 (2.5-fold), NAGT I (4-fold) and Mann all cells as described by Nilsson et al. (1994). Expressed II (6-fold). At the higher end was SialylT which was expressed protein was detected using either specific polyclonal antibodies 10-fold over endogenous levels in the TGN38/SialylT-HeLa or monoclonal antibodies to an epitope tag engineered onto the cell line and 50-100-fold in the SialylT-HeLa cell line. Inter- C-terminus of the enzyme (Table 1). The structure and estingly the difference in expression levels between these two topology of the proteins under study is summarised in Fig. 1. cell lines had no effect on the distribution of the protein within Stable HeLa cell lines expressing NAGT I (Nilsson et al., the Golgi apparatus. As shown in Table 2, 68% of the SialylT 1993) and Mann II (Nilsson et al., 1994) have been charac- was present in the TGN in the TGN38/SialylT-HeLa cell line terised previously. Cells expressing SialylT either alone or compared with 70% in the SialylT-HeLa cell line. Similar together with TGN38 were characterised by western blotting. results were also obtained when the Mann II-HeLa cell line As shown in Fig. 2 (lanes 1 and 2), Golgi membranes from was compared with another clone expressing the protein at a either cell line expressed a single protein of 54 kDa. This is 3-fold lower level (2-fold over NRK cells) (data not shown). higher than the 47 kDa reported for the protein from rat liver The effect of expressed proteins on endogenous Golgi proteins (Weinstein et al., 1987) and presumably reflects increased gly- was determined by measuring the distribution of endogenous cosylation in HeLa cells. The converse was true for TGN38 in GalT within the Golgi apparatus by immuno-gold microscopy. the TGN38/SialylT-HeLa cells. A single protein of 58 kDa was In the parental cell line, 33±14% of the GalT was present in the expressed (Fig. 2, lane 3), lower than the 85-95 kDa reported stack, the rest in the TGN. As shown in Table 2 this was not for the heterogeneously sialylated protein from NRK cells changed significantly by stable expression of any of the proteins. (Luzio et al., 1990). The level of GalT in the stack in the stable cell lines ranged from Immunofluorescence microscopy of these two cell lines also 28 to 34%. Furthermore, the level of GalT in each of the stable gave the expected pattern. SialylT was localised to a compact cell lines was not affected by expression of any of the other reticulum on one side of the nucleus in both SialylT-HeLa cells proteins since the total number of gold particles/Golgi apparatus (Fig. 3A) and TGN38/SialylT-HeLa cells (data not shown). only varied between 34 and 40 (Table 2). TGN38 was present in the same structure but also in punctate structures throughout the cell cytoplasm which likely represent Immuno-gold microscopy peripheral endosomes (Ponnambalam et al., 1994) (Fig. 3B). An extensive series of experiments was carried out to establish the distribution of the four Golgi enzymes and TGN38. One of Table 2. Distribution of GalT and Sialyl T between the them, GalT was used as the reference marker for each of the Golgi stack and the TGN in different cell lines others. We had earlier shown that the distribution of NAGT I Percentage overlapped that of GalT (Nilsson et al., 1993); Mann II was distribution found to overlap the distribution of GalT in the exactly same of gold particles way (Fig. 4A) showing, by inference, that it had the same dis- Total number of (%) tribution as NAGT I. In contrast, Sialyl T had exactly the same gold particles/ Cell line Enzyme Golgi apparatus Golgi stack TGN distribution as GalT (Fig. 4B). To provide a more quantitative measure of the distribution, Parental-HeLa GalT 35±15 33±14 67±14 Mann II -Hela GalT 40±19 31±13 69±13 we performed single labelling of all the cell lines and applied NAGT1-HeLa GalT 38±17 33±15 67±15 a Stereological method described earlier (Nilsson et al., 1993) SialylT-HeLa GalT 36±21 28±18 72±18 in which the trans-most, or T cisterna of the stack was defined SialylT 44±30 30±10 70±10 as the last continuous cisterna on the side of the stack that TGN38/SialylT-HeLa GalT 34±11 34±14 66±14 labelled for GalT. This cisterna was used as the reference SialylT 3±1.5 32 68 point for all other compartments. The T-1 and T-2 cisternae Cryo-sections from the different cell lines were labelled for either GalT or most likely represent the medial and cis cisternae, respec- SialylT. Gold particles (350 to 800) over 10 to 20 Golgi apparatus were tively. This is because there are typically three cisternae in the counted and the results expressed either as the total or as the percentage over stack in HeLa cells (Nilsson et al., 1993). The T-3 compart- the Golgi stack or TGN ± s.e.m. In the case of SialylT in the TGN38/SialylT-HeLa cell line the number of ment likely represents the CGN but was not readily identified gold particles was too few to permit an estimation of the s.e.m. In this case in many cross sections and was, for the most part, unlabelled the distribution of 78 gold particles in 30 cells from two experiments was for any of the Golgi proteins under study. It was not consid- determined. ered further. Mapping the distribution of resident Golgi proteins 1621

Fig. 4. Distribution of Mann II/GalT and SialylT/GalT by double label immunogold microscopy. Thin frozen sections of (A) Mann II- Hela or (B) SialylT- HeLa cells were double- labelled so as to reveal the location of (A) Mann II (15 nm Protein A gold) and GalT (10 nm Protein A gold); (B) GalT (goat anti-rabbit coupled to 5 nm gold) and SialylT (goat anti- mouse coupled to 10 nm gold). In A, note that GalT is present in the TGN (asterisk) and the trans cisterna, overlapping the distribution of Mann II, which is found in the trans and medial cisternae. In contrast, in B, GalT co-distributes with SialylT, being present in both the trans cisterna and the TGN (asterisk). The primary antibodies are listed in Materials and Methods and Table 1. Tubular extensions (small arrows, Klumperman et al., 1993) of vacuolar (E) are not labelled. They can be distinguished from the COP-coated vesicles (large arrows, Oprins et al., 1993) due to their denser content and their lack of coat. N, nuclear envelope; M, ; G, Golgi cisternae. Bar, 200 nm. 1622 C. Rabouille and others Mapping the distribution of resident Golgi proteins 1623

The T+1 compartment represents the TGN which is a pleo- elements that emanate a considerable distance from the stack. morphic structure comprising flattened, cisternal elements This structure was quantitated as described in Materials and abutting the trans cisterna linked to extensive tubulo-reticular Methods.

Fig. 5. Distribution of the stably expressed proteins by single label immunogold microscopy. Thin frozen sections of the stable HeLa cell lines listed in Table 1 were labelled so as to reveal the location of: (A) NAGT I (goat anti-mouse coupled to 10 nm gold); (B) Mann II (10 nm Protein A gold); (C) SialylT (goat anti-mouse coupled to 10 nm gold); and (D) TGN38 (goat anti-rabbit coupled to 10 nm gold). Note that in A and B, the gold is mainly restricted to the stacked Golgi cisternae (G) whereas, in C and D, the vast majority is over the TGN (asterisks). The primary antibodies are listed in Materials and Methods and Table 1. E, vacuolar endosome; ER, ; M, mitochondrion; N, nuclear envelope. Bars, 200 nm. 1624 C. Rabouille and others

Distribution of NAGT I and Mann II compartments (Fig. 5A). More than 65% of the labelling (Fig. Labelling for NAGT I was restricted almost exclusively to the 6A) was present over two adjacent cisternae on the trans side Golgi stack with little label over the TGN or other membrane of the stack (Nilsson et al., 1993). Mann II was also present over two cisternae on one side of the stack (Fig. 5B) which was shown to be the trans side by double-labelling for GalT (Fig. 4A). More than 75% of the labelling was present over the medial and trans cisternae (Fig. 6A). The linear density of labelling for both NAGT I and Mann II in the medial and trans cisternae was at least 2 times higher than in the cis cisterna and 5-7 times higher than in the TGN (Fig. 6B). Distribution of GalT and SialylT Labelling for both SialylT (Fig. 5C) and GalT was restricted to the trans cisterna and the TGN. There was little labelling over the medial or cis cisternae. About 20% of labelling for GalT and SialylT was present in the trans cisterna and about 70% in the TGN (Fig. 6A). The co-distribution of these two enzymes was confirmed by double-labelling SialylT-HeLa cells for both GalT and SialylT (Fig. 4B). Quantitation showed that the linear density of both GalT and SialylT in the trans cisterna was about 4.5 times that in the medial cisterna and about 20 times that in the cis cisterna (Fig. 6B). The average linear density in the TGN was lower than that in the trans cisterna even though it contained about 70% of both enzymes. This is because the TGN has a much longer membrane length. Distribution of TGN38 TGN38 was originally described as a marker for the TGN in NRK cells (Luzio et al., 1990) and, when expressed either tran- siently at low levels (Ponnambalam et al., 1994) or stably (Fig. 5D) in HeLa cells, it is also present almost exclusively in the TGN. As shown in Fig. 6A, fully 90% of TGN38 was present in the TGN in the TGN38/SialylT-HeLa cell line. The linear density in the TGN was 7 times higher than that in the trans cisterna and 30 times higher than in the medial cisterna. None could be detected in the cis cisterna (Fig. 6B).

DISCUSSION

The work described in this paper both confirms and extends our earlier observations on the overlapping distribution of NAGT I and GalT (Nilsson et al., 1993) to include both Mann II and SialylT. The four enzymes fell into two groups: NAGT I co-distributed with Man II whereas GalT co-distributed with SialylT. The overlap was in the trans cisterna which had all four enzymes. Stable cell lines As before, considerable care was taken to ensure that the stably expressed protein had the same distribution in HeLa cells as the endogenous protein and did not alter the distribution of Fig. 6. Quantitative distribution of GalT and the stably expressed other Golgi enzymes. First, the origin of the cDNAs was either proteins. Proteins were localised as described in Table 2 and Fig. 4 and the distribution of gold particles over the TGN, T (trans), T-1 human (NAGT I, SialylT) or a closely related mammal (Mann (medial) and T-2 (cis) cisternae was determined and expressed either II - murine, TGN38 - rat). The sequence similarity between as a relative distribution (A) or a linear density (B). 250-1200 gold Golgi enzymes from different mammals is typically in excess particles were counted over 15-20 Golgi apparatus in two separate of 90% (for review, see Kleene and Berger, 1993). Second, the experiments and two grids and the results are presented as the mean epitope tag, when present, was placed at the C-terminus, as far ± s.e.m. away as possible from the membrane-spanning domain that Mapping the distribution of resident Golgi proteins 1625 contains the signal for retention (for references, see Machamer, unclear but one possibility relates to the recent finding that 1993). Third, the level of expression was in all but one case selective re-distribution of NAGT I and Mann II from the less than or equal to 10 times the level of the endogenous Golgi to the ER resulted in the disappearance of the Golgi stack protein. Since none of the Golgi enzymes constitute more than (Nilsson et al., 1994). This strongly suggests that these a few per cent of Golgi membrane, such an increase could rea- enzymes (and perhaps others in the same cisternae) are sonably be expected to have a minimal effect on the distribu- involved in maintaining the structure of the stack. A simple tion of the protein. In fact, the one exception showed that con- model would be for these enzymes to interact with each other siderable over-expression had no effect on the distribution. across the intercisternal space which means that they would SialylT was expressed at 10 times the endogenous level in have to be present in adjacent cisternae. The overlapping dis- TGN38/SialylT-HeLa cells but at 50-100 times in SialylT- tribution of Golgi enzymes would, therefore, reflect the HeLa cells, yet the distribution of SialylT between the trans stacking mechanism of Golgi cisternae. A simple functional cisterna and the TGN was almost exactly the same in both cell consequence of this arrangement is that transported proteins lines (Table 1). Lastly, the distribution of GalT was checked would meet the same set of enzymes twice. If they failed to be in each of the stable cell lines. The results showed clearly that modified the first time, there would be a second chance. none of the stably expressed proteins affected the distribution Though the majority of enzyme was present in two adjacent of at least this one Golgi protein. compartments there was often significant amounts in the flanking cisternae. One possibility is that these enzymes Co-distribution of enzymes represent that portion of the protein being recycled. No The co-distribution of NAGT I and Mann II is in agreement retention mechanism is perfect and enzymes might be expected with earlier work. Relocation of NAGT I to the ER by attach- to leak into transport vesicles. These might then be recycled as ment of an ER retrieval signal causes accumulation of Mann II has been shown for both soluble and membrane proteins of the in the ER, and relocation of Mann II has a similar effect on ER (for review, see Nilsson and Warren, 1994). There are NAGT I pointing to a very specific association between these several Golgi proteins that appear to recycle though the two enzymes (Nilsson et al., 1994). When rat liver Golgi stacks retrieval signal has not been identified (Alcalde et al., 1994; are extracted with Triton X-100, most of the NAGT I and Mann Johnston et al., 1994). Another possibility is that overlapping II remain in the Triton pellet whereas most of the markers from enzymes interact weakly with each other. As an example, relo- other parts of the Golgi are released into the supernatant cation of NAGT I to the ER not only caused re-location of (Slusarewicz et al., 1994). Even earlier work showed that Mann II but also some of the GalT (Nilsson et al., 1994). The NAGT I and Mann II co-fractionated on sucrose gradients as presence of some GalT and SialylT in the medial cisterna did GalT and SialylT, but at a lower density of sucrose (Dunphy might then be explained by such an interaction. One last pos- and Rothman, 1983; Goldberg and Kornfeld, 1983). Interest- sibility is that the distribution is the consequence of a retention ingly, the two sets of peaks overlapped in agreement with the mechanism based on the increasing thickness of the lipid overlap of all four enzymes in the trans cisterna. At that time bilayer through the Golgi stack (Bretscher and Munro, 1993). the aim was to show that Golgi enzymes were in separate com- Golgi enzymes would move until the length of the spanning partments so the overlap was either ignored or put down to a domain matched the thickness of the bilayer. The increment in limitation in the resolution of the technique. thickness might, however, be sufficiently small to permit accu- The co-distribution of GalT and SialylT agrees with most mulation in several adjacent and flanking cisternae. but not all published work. Though early biochemical studies showed that both enzymes co-fractionated on sucrose gradients Conclusions (Dunphy and Rothman, 1983), studies on recycling proteins Continued mapping of the Golgi apparatus in HeLa cells has suggested that they were in different compartments (Duncan strengthened the idea that it is a precisely defined structure. and Kornfeld, 1988; Huang and Snider, 1993). This discrep- The Golgi enzymes involved in the construction of complex, ancy might reflect the fact that CHO cells were used for these N-linked oligosaccharides are not restricted to single cisternae experiments and there is evidence in this cell line that GalT but, so far, they are restricted to two adjacent ones. The over- and SialylT are in different compartments (Chege and Pfeffer, 1991). It will be important to confirm this using the approach lapping distribution may reflect the structural organisation of we have used for HeLa cells. the stack. It is, however, clear that the distribution of Golgi Earlier microscopic studies, both immuno-gold and enzymes does vary from tissue to tissue and from organism to immunofluorescence, also suggested that GalT and SialylT organism (Roth et al., 1986; Velasco et al., 1993). This were present in different compartments (see Berger et al., 1993, suggests that the mechanism governing the localisation of for references) but this discrepancy was due, at least in part, to Golgi enzymes is subject to another layer of control. The most the use of antibodies that recognised oligosaccharides as well obvious would be post-translational modifications such as as the enzyme polypeptide chain. The most recent immunoflu- phosphorylation, and work is presently underway to test this orescence data show that GalT and SialylT co-localise almost possibility. entirely at the immunofluorescence level (Berger et al., 1993) We are indebted to Dr Paul Luzio (Cambridge, UK) and Dr George and the data presented in this paper, also using antibodies to Banting (Bristol, UK) for kindly providing us with the cDNA and the polypeptide chain of GalT, show exact co-localisation by antibodies for TGN38; Dr Thomas Kreis (Geneva, Switzerland) for high resolution immuno-gold microscopy. the P5D4 hybridoma; Dr Kelley Moremen (Athens, Georgia) for Mann II polyclonal antibodies; and Dr Sean Munro (Cambridge, UK) Mechanism of overlap for cDNA encoding SialylT. We also thank the oligosynthesis facility The mechanism that generates the overlapping distribution is at Clare Hall for high quality oligonucleotides; the photography 1626 C. 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