(Received for publication, February 10, 1995; and in revised form, July 27, 1995)
From the
ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc-GlcNAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-GlcNAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.
Transport from the endoplasmic reticulum (ER) ()to
the cell surface occurs by default. No signals are needed for proteins
to move from one compartment to the next along the exocytic pathway
(Pfeffer and Rothman, 1987; Pryer et al., 1992; Rothman and
Orci, 1992). Signals are needed to divert proteins in the trans Golgi network (TGN) to organelles such as the lysosomes (Kornfeld
and Mellman, 1989) and secretory granules (Bauerfeind and Huttner,
1993). They are also needed to localize proteins to particular
compartments on the exocytic pathway. These signals are of two types:
retention signals and retrieval signals (Nilsson and Warren, 1994).
Retention signals provide the primary means of localizing a protein to
a particular compartment. The best characterized are those located in
the membrane-spanning domain (and flanking sequences) of Golgi enzymes,
though the precise manner in which they work has still to be elucidated
(Bretscher and Munro, 1993; Nilsson et al., 1993b). Retrieval
signals have been identified on soluble proteins in the ER (Munro and
Pelham, 1987) and on membrane proteins both in the ER (Nilsson et
al., 1989; Jackson et al., 1993; Schutze et al.,
1994) and the TGN (Bos et al., 1993; Humphrey et al.,
1993; Wong and Hong, 1993). These function to return proteins to their
correct compartments after they have left them. The act of leaving may
reflect the limitations of the retention mechanism in which case
retrieval is a salvage process (Warren, 1987). Alternatively, it may
reflect the transport process, which requires reusable proteins to
package and deliver cargo to the next compartment. In this case,
retrieval is a recycling process (Pelham, 1989).
In one case, that of TGN38 (Ponnambalam et al., 1994), a retention signal has been identified in the membrane-spanning domain and a retrieval signal in the cytoplasmic tail. This suggests that all proteins along the exocytic pathway might be equipped in this manner, with the membrane-spanning domain providing the primary means of localizing the protein and the retrieval signal acting to return the protein should it either deliberately or accidentally leave the compartment in which it mostly resides.
Retrieval signals have not yet been identified in resident Golgi enzymes, though there is some evidence that they are retrieved from later compartment(s). Mannosidase II, for example, is present in medial/trans cisternae in most cells (Velasco et al., 1993), yet the bound oligosaccharides are modified by sulfate residues (Moremen and Touster, 1985), a modification that is restricted to the TGN (Baeuerle and Huttner, 1987). Other Golgi proteins have also been shown to contain oligosaccharides that suggest passage beyond the point at which they normally reside (Yuan et al., 1987; Gonatas et al., 1989; Johnston et al., 1994; Alcalde et al., 1994). It is, however, possible that these modifications are caused not by recycling but by the presence of small amounts of the oligosaccharide-modifying enzymes in compartments other than those in which they mostly reside. We have shown recently that enzymes involved in the construction of complex N-linked oligosaccharides are mostly present in two adjacent cisternae, but small amounts are present in flanking cisternae (Nilsson et al., 1993a).
We set out to devise a system that would measure the retrieval of a resident Golgi enzyme. We exploited the properties of a Chinese hamster ovary cell line (ldlD) generated by Krieger and colleagues (Kingsley et al., 1986), which lacks the epimerase activity needed to synthesize Gal and GalNAc from glucose precursors. The consequence is that synthesis of O-linked oligosaccharides cannot be initiated, and N-linked oligosaccharides are truncated at the point where terminal Gal would have been added. These defects are rapidly reversed by the addition of exogenous Gal and GalNAc.
The synthesis of O-linked oligosaccharides is initiated in the CGN (Tooze et al., 1988; Niemann et al., 1982; Krijnse-Locker et al., 1994) and the cis cisterna of the Golgi stack (Roth, 1984; Deschyyteneer et al., 1988; Roth et al., 1994) by the addition of GalNAc to serine or threonine residues. The CGN (Huttner and Tooze, 1989; Hsu et al., 1991; Pelham, 1989), also known as the intermediate (Saraste and Kuismanen, 1984; Schweizer et al., 1990) or salvage compartment (Warren, 1987), receives the entire output of newly synthesized proteins from the ER and delivers them to the cis cisterna of the Golgi stack. This includes newly synthesized enzymes that reside in the Golgi stack. We focused on GlcNAc-T I, both because it resides in the medial/trans cisternae in most cells (Velasco et al., 1993) and it contains only O-linked and not N-linked oligosaccharides (Kumar et al. (1990) and this paper), a feature that considerably simplified analysis.
We reasoned that, in the absence of exogenous GalNAc and Gal, GlcNAc-T I, lacking O-linked oligosaccharides, would be delivered to the Golgi stack. Evidence in support of this assertion comes from the kinetics of maturation of the human respiratory syncitial virus in ldlD cells (Wertz et al., 1989). In the absence of exogenous GalNAc and Gal, the bound oligosaccharides of the human respiratory syncitial virus G protein are chased into an endoglycosidase H-resistant form. This can only occur if GlcNAc-T I is active and located in the Golgi apparatus. After delivery of the unglycosylated GlcNAc-T I to the Golgi stack, Gal and GalNAc would be added to restore the synthesis of O-linked oligosaccharides. If GlcNAc-T I recycled and the salvage pathway involved passage back through the CGN and the cis cisterna, GalNAc would be added, followed by Gal and sialic acid once the enzyme reached its original location in the medial/trans part of the stack. Here, we show that GlcNAc-T I did acquire these sugars and that this was prevented by inhibitors of vesicle-mediated traffic.
Sub-confluent monolayers (60%) of parental ldlD cells were transfected with 20 µg of cDNA encoding Myc-tagged GlcNAc-T I. Stable clones were selected and then maintained in the presence of geneticin (400 µg/ml; Life Technologies, Inc.).
Cycloheximide (Sigma) was added to a final concentration of 30 µg/ml, 4 h before addition of exogenous GalNAc and Gal, and was then present throughout the remainder of the experiment in all washes and chase medium. The sugars (GalNAc and Gal, both from Sigma), when present, were added to a final concentration of 400 µg and 20 µg/ml, respectively, for up to 4 h.
For NEM treatment, cells were treated with cycloheximide for 3.5 h, washed with ice-cold Hank's buffered saline solution, incubated with 1 mM NEM in the same buffer for 15 min at 0 °C, and washed; excess NEM was quenched at room temperature for 5 min using 2 mM dithiothreitol. After two more buffer washes, incubation was continued in ITS medium in the presence or absence of GalNAc/Gal for 4 h.
For treatment with okadaic acid, cells were treated with cycloheximide for 3 h before addition of 1 µM okadaic acid for 1 h. Incubation was then continued in the presence or absence of GalNAc/Gal for 4 h.
To determine the labeling density, the
sectioned surface area of Golgi stacks, the Golgi area, or the nucleus
was determined by a point-hit method (Weibel et al., 1969)
using the formula A = (Pd
)/M
, where A = the sectioned surface area, P = the
grid intersections falling over the area, d = the
distance between grid lines, and M = the final
magnification of the photograph.
The number of gold particles associated with the measured area was counted, and the density was determined by N/A, with N being the number of gold particles. For each sample, at least 15 Golgi areas from cells from two experiments were counted, and the results are presented as the mean ± S.D.
This modified cDNA was subcloned into the pSR vector, and
stable transfectants were selected in the presence of geneticin
(Southern, 1981; Canaani and Berg, 1982). Mixed clones were used in
which more than 90% of the cells expressed similar levels of protein as
assessed by immunofluorescence microscopy.
To determine the composition of the O-linked oligosaccharides on the Myc-GlcNAc-T I, cells were grown for 3 days in McCoy's medium supplemented with insulin, transferrin, and selenium (ITS medium) in the presence or absence of GalNAc/Gal or in the presence of FCS. After lysis and fractionation by SDS-PAGE, total membrane proteins were transferred to membranes and were probed with the monoclonal antibody 9E10.
Three forms of the protein were detected. An unglycosylated
form of 51 kDa, an immature form of 52 kDa, and a mature form of 55
kDa. The unglycosylated form of 51 kDa was the major form of the
protein found in cells grown for 3 days in ITS medium alone (Fig. 1A, lane1). The molecular mass
of 51 kDa is very similar to that calculated from the sequence of the
protein, 50.9 kDa (Kumar et al., 1990). A minor form of 52 kDa
was also present. The amount of the minor 52-kDa form increased with
time in ITS medium. Since this form migrated almost exactly with that
obtained by incubating the cells in ITS containing GalNAc (Fig. 1A, lane2), it seemed likely
that it represented an immature form of the protein (see
``Discussion''). Sugars such as GalNAc can sometimes be
salvaged during degradation of endogenous proteins (Krieger et
al., 1989). When cells were incubated in ITS medium containing
both GalNAc and Gal, the molecular mass of Myc-GlcNAc-T I increased to
55 kDa (Fig. 1A, lane3), the same as
that obtained in the presence of dFCS (Fig. 1A, lane4). These mature forms of Myc-GlcNAc-T I were
also sensitive to treatment with neuraminidase (Fig. 1B, lanes1-4), showing
that the O-linked oligosaccharides contained sialic acid.
Taken together, these data show that a mixture of GalNAc and Gal is
both necessary and sufficient for the construction of the O-linked oligosaccharides found on the mature protein.
Furthermore, the difference in molecular mass between the
unglycosylated and mature forms of the protein (4 kDa) is
consistent with the addition of two simple O-linked
oligosaccharides (Kornfeld and Kornfeld, 1980).
Figure 1: Composition of O-linked oligosaccharides bound to Myc-GlcNAcT I in ldlD cells. A, cells grown in complete growth medium for 12 h were transferred to ITS alone (lane1), ITS containing GalNAc (lane2), ITS containing GalNAc and Gal (lane3), or in ITS containing dFCS (complete growth medium, lane4). After 3 days, cell extracts were fractionated by SDS-PAGE and Western blotted using the 9E10 antibody to detect the Myc-GlcNAcT I. The protein bracketedbydots in lane2 was only present in some experiments and may represent the production of sugars by a salvage pathway. B, after 3 days in ITS containing GalNAc/Gal (lanes1 and 2) or dFCS (lanes3 and 4), cell lysates were either mock-treated (lanes1 and 3) or treated (lanes2 and 4) with neuraminidase before fractionation by SDS-PAGE and Western blotting. Molecular masses on the left indicate Myc-GlcNAc-T I containing no sugars (51 kDa), GalNAc (52 kDa), GalNAc/Gal (54 kDa), or GalNAc/Gal/sialic acid (55 kDa).
Table 1also
shows that the specific activity of GlcNAc-T I was unaffected by the
addition of GalNAc/Gal to the ITS growth medium. Since this treatment
adds O-linked oligosaccharides to pre-existing Myc-GlcNAc-T I
(see below and Fig. 4), it is clear that these O-linked
oligosaccharides have no significant effect on the catalytic activity
of the protein. Similar results were obtained for
1,4-galactosyltransferase.
Figure 4: Time course of acquisition of O-linked oligosaccharides. A, after 3 days growth in ITS medium, cycloheximide was added, followed 4 h later by GalNAc/Gal for up to another 4 h. Cell extracts were fractionated by SDS-PAGE, Western blotted using the 9E10 antibody to detect the Myc-GlcNAc-T I (top), and the lanes scanned so as to quantitate the conversion of the unglycosylated and immature forms of Myc-GlcNAc-T I (51- and 52-kDa forms) to the mature form (55 kDa). B, cells incubated for 4 h in the absence (lanes1 and 2) or presence (lanes3 and 4) of GalNAc/Gal were either mock-treated (lanes1 and 3) or treated (lanes2 and 4) with neuraminidase before fractionation by SDS-PAGE and Western blotting.
A small but significant increase in the specific activities of both enzymes in both cell lines when the cells were grown in ITS medium containing dFCS likely reflects the presence of growth factors that increase the growth rate.
When the stably transfected cells were grown under optimal conditions, in the presence of ITS and dFCS, GlcNAc-T I was localized by immunofluorescence to a compact juxtanuclear reticulum characteristic of the Golgi apparatus in these cells (Fig. 2A). This location was confirmed by double labeling for the resident enzyme, mannosidase II (Louvard et al., 1982; Velasco et al., 1993) (Fig. 2B). Exactly the same pattern was obtained when the cells were grown in the presence of ITS medium alone in the absence (Fig. 2, C and D) or presence (Fig. 2, E and F) of GalNAc/Gal. The recycling experiments described below had to be conducted in the presence of cycloheximide, and it was, therefore, important to show that inhibition of protein synthesis had no effect on the distribution of Myc-GlcNAc-T I. Treatment with cycloheximide alone for 8 h had no effect (data not shown) and neither did a 4-h treatment continued for a further 4 h in the presence of GalNAc/Gal (Fig. 2, G and H). In addition, no Myc-GlcNAc-T I was detected to be secreted into the medium (data not shown) under all conditions.
Figure 2:
Localization of Myc-GlcNAc-T I in ldlD
cells by immunofluorescence microscopy. Cells were grown for 3 days in
either ITS containing dFCS (panelsA and B)
or ITS alone (panelsC and D). The latter
were then treated for 4 h with GalNAc/Gal (panelsE and F) or for 8 h with cycloheximide (CHX), the
last 4 h of which were in the presence of GalNAc/Gal (panelsG and H). Cells were then fixed, permeabilized,
and double-labeled for Myc-GlcNAc-T I (panelsA, C, E, and G) and mannosidase II (MannII, panelsB, D, F,
and H). Magnification, 60.
These results were
both confirmed and extended by immunogold microscopy on cryosections.
For cells grown in the presence of dFCS alone (Fig. 3A)
or in the presence of GalNAc/Gal with (Fig. 3D) or
without (Fig. 3C) cycloheximide, the results were
indistinguishable. Labeling was restricted to stacked Golgi cisternae,
and between two and four adjacent cisternae were labeled. There was no
significant labeling over any other structure in the cell. Quantitation
showed that the level of labeling was also very similar, the average
labeling density over Golgi stacks only varying between 24 and 28 gold
particles/µm (Table 2).
Figure 3: Localization of Myc-GlcNAc-T I in ldlD cells by immunoelectron microscopy. Cells were grown for 3 days in either ITS containing dFCS (panelA) or ITS alone (panelB). The latter was then treated for 4 h with GalNAc/Gal (panelC) or treated for 8 h with cycloheximide, the last 4 h of which were in the presence of GalNAc/Gal (panelD). Cells were then fixed, frozen, sectioned, and labeled with 9E10 antibodies followed by rabbit anti-mouse antibody coupled to 10 nm of gold. The inset in panelB shows the Golgi stacks found in about 30% of cells. Bar, 0.25 µm. Nu, nucleus, G, Golgi.
The only exception to this distribution was found in transfected cells grown in the absence of any additional components, that is, in the presence of ITS medium alone. In these cells, the Golgi membranes appeared to be less cisternal and more tubulo-reticular (Fig. 3B). Golgi stacks were present (inset to Fig. 3B) in about 30% of the cells. The density of gold particle labeling over the Golgi stack was only just over half that of the other samples (Table 2). However, this did not mean that Myc-GlcNAc-T I had been lost from the Golgi apparatus because the density of labeling over the entire Golgi region, including the tubular and vesicular structures closely associated with the stacks, was very similar for all the samples (Table 2). The glycosylation state of Golgi proteins may, therefore, affect the morphology of the stack, but it did not change the level or Golgi location of Myc-GlcNAc-T I.
Addition of GalNAc/Gal resulted in the disappearance of the unglycosylated form of the protein and the appearance of the immature form. This was followed by the appearance of the mature form with a half-time of about 150 min (Fig. 4A). Treatment with neuraminidase showed that the mature form had acquired sialic acid residues (Fig. 4B). In the absence of GalNAc/Gal, the levels of unglycosylated and immature forms of the protein were generally unaffected over the time of incubation, although occasionally a slight increase in the immature form was noted (data not shown).
Since the acquisition of mature O-linked oligosaccharides was not accompanied by any change in the steady state localization of Myc-GlcNAc-T I (Fig. 3), this suggested that the Myc-GlcNAc-T I was undergoing retrograde transport to the CGN and anterograde transport back to the Golgi stack. If true, then this process should be sensitive to inhibitors of vesicular transport. NEM inhibits the fusion of transport vesicles with their target membranes (Glick and Rothman, 1987), and, as shown in Fig. 5(lanes3 and 4), it almost completely abolished the formation of mature Myc-GlcNAc-T I. Okadaic acid caused vesiculation of the Golgi apparatus and an inhibition of intracellular transport (Lucocq et al., 1991). It also abolished the formation of mature Myc-GlcNAc-T I (Fig. 5, lanes5 and 6).
Figure 5: Inhibition of vesicular transport prevents the addition of O-linked oligosaccharides. After growth for 3 days in ITS medium, cycloheximide was added followed by either mock treatment (lanes1 and 2) or treatment with NEM (lanes3 and 4) or okadaic acid (lanes5 and 6). The cells were then chased for 4 h in the absence (lanes1, 3, and 5) or presence (lanes2, 4, and 6) of GalNAc/Gal. The molecular masses on the left indicate the unglycosylated (51 kDa) and mature (55 kDa) forms of Myc-GlcNAc-T I. A doublet was reproducibly obtained in the presence of NEM, but the identity of the lower molecular mass form is unclear.
There are two assumptions implicit in the experiments just described. The first is that GlcNAc-T I, lacking O-linked oligosaccharides, is correctly assembled and transported to the Golgi stack. There is evidence to suggest that this is the case (Kozarsky et al., 1988; Wertz et al., 1989), but a more rigorous demonstration was deemed necessary because a modified version of GlcNAc-T I had to be used, since antibodies were not available to the endogenous protein.
GlcNAc-T I was chosen because it possesses
only one type of oligosaccharide, which simplifies analysis, and
because O-linked oligosaccharides are added
post-translationally, after or during the latter stages of the folding
process. This minimizes the possibility that GlcNAc-T I will fold
incorrectly in the absence of GalNAc and Gal and be rapidly degraded.
The suitability of this choice was confirmed by pulse-chase (data not
shown) and Western blotting experiments, which estimated the half-life
of the unglycosylated and mature proteins at about 10 h, not too
dissimilar from the 10-20 h that characterizes the half-life of
other Golgi proteins (Strous and Berger, 1982; Yuan et al.,
1987). More importantly, the transfected cells were shown to have
10 times the activity of GlcNAc-T I when compared to the parental
cell line. Catalytic activity is probably the most sensitive measure of
correct folding. The specific activity of Myc-GlcNAc-T I was also not
affected by the absence of O-linked oligosaccharides, arguing
strongly that they play no role in correct assembly or functioning of
the protein.
The location of Myc-GlcNAc-T I was assessed by both immunofluorescence and immunoelectron microscopy. Myc-GlcNAc-T I was localized to a compact juxtanuclear reticulum that characterizes the Golgi apparatus in animal cells (Louvard et al., 1982). The protein also colocalized exactly with an endogenous Golgi marker, mannosidase II. At the electron microscopic level, the protein was found exclusively in the Golgi region. There was no significant labeling over the ER or plasma membrane.
Though both forms of Myc-GlcNAc-T I were present in the Golgi region, it was clear that the lack of sugars changed the morphology of the Golgi stack. Stacked cisternae were only present in around 30% of the cells, but in all the rest of the cells, the Golgi was a more tubulo-reticular structure, suggesting that the oligosaccharides bound to resident proteins might play a structural role in the organization of the Golgi stack. The simplest idea is to suggest that they contribute toward a lumenal Golgi matrix that serves to keep the central portions of cisternae apart. In their absence, apposing cisternal membranes would touch, thereby triggering periplasmic fusion and the formation of a tubulo-reticular structure. These ideas are more fully explored in a recent review (Rothman and Warren, 1994). For the present purposes, it is sufficient that the Myc-GlcNAc-T I is restricted to the Golgi complex region and physically separated from the GalNAc-T that initiates synthesis of O-linked oligosaccharides.
The physical separation of Myc-GlcNAc-T I and GalNAc-T is the second assumption implicit in these experiments. Most work places GlcNAc-T I in the medial/trans cisternae and GalNAc-T in the CGN, though some recent data show that GalNAc-T can be present in the cis cisterna in some cells (Roth et al., 1994). Golgi enzymes are mostly found in two adjacent cisternae, but small amounts are found in flanking cisternae. If this is true for GlcNAc-T, then the results could be explained by the presence of small amounts of this enzyme in medial/trans cisternae. Addition of GalNAc/Gal could have led to slow maturation of Myc-GlcNAc-T I without the need for it to recycle via the CGN.
To demonstrate that this was not occurring and that the maturation of Myc-GlcNAc-T I required vesicular transport, we investigated the effects of several transport inhibitors. Both anterograde (Rothman, 1994) and retrograde (Letourner et al., 1994) transport through the Golgi stack is mediated by COP I-coated vesicles, which bud from one cisterna and fuse with the next on the pathway. Fusion of these vesicles with their target membrane can be inhibited by pretreatment with NEM, which inactivates the general fusion protein, NSF (NEM-sensitive factor) (Glick and Rothman, 1987). Fusion is also inhibited during mitosis (Misteli and Warren, 1994) and leads to the fragmentation of the Golgi apparatus (Warren, 1993). Fragmentation of the Golgi apparatus can be mimicked by the addition of okadaic acid (Lucocq et al., 1991).
Both NEM and okadaic acid completely prevented the maturation of Myc-GlcNAc-T I. It is, of course, possible that these drugs affected the enzymes (and sugar transporters) involved in the oligosaccharide maturation process, but this seems unlikely given the differences in their chemical structure and mode of action. NEM acts on thiol residues, whereas okadaic acid specifically (and non-covalently) inhibits protein phosphatases 1 and 2A (Cohen et al., 1990). Furthermore, NEM has been shown to have no effect on the processing of both N- and O-linked oligosaccharides bound to either proteins (Glick and Rothman, 1987; Rothman, 1987) or glycolipids (Wattenberg, 1990). Okadaic acid has been shown to have no effect on the addition of dolichol oligosaccharides to newly synthesized proteins in the ER (Lucocq et al., 1991). Taken together, these data suggest that the observed addition of O-linked oligosaccharides to Myc-GlcNAc-T I is the consequence of recycling via the CGN.
The half-time for recycling of Myc-GlcNAc-T I as measured
by the acquisition of O-linked oligosaccharides was about 150
min. Newly synthesized Myc-GlcNAc-T I matured with a half-life of
15-20 min (data not shown), and other work has shown that about
half of this time is usually involved in anterograde transport through
the Golgi stack (Green et al., 1981). This means that the
half-time for retrograde transport to the CGN was about 140 min. At
first sight, this is inconsistent with the conversion of unglycosylated
Myc-GlcNAc-T I to the immature form, the half-time of which was about
5-10 min. There is, however, evidence to suggest that there is an
additional unglycosylated form of Myc-GlcNAc-T I, which has almost the
same molecular mass as the immature form (52 kDa). Despite intensive
efforts by us and others, ()we have not been able to define
the modification that generates this other form, but it does more
readily explain why most incubations in the absence of GalNAc/Gal
yielded two forms of Myc-GlcNAc-T I.
In summary, we have shown that unglycosylated Myc-GlcNAc-T I resides in the Golgi in ldlD cells starved of GalNAc/Gal. Upon addition of these sugars, the protein acquires O-linked oligosaccharides with a half-time of several hours. Acquisition of the sugars is prevented by inhibitors of vesicular traffic, suggesting that the protein is transported first to the CGN and then back to the Golgi stack. Further work is still needed to confirm a role for vesicle-mediated transport, but this system should provide a convenient means of studying recycling pathways within the Golgi apparatus.