From the Imperial Cancer Research Fund, Cell Biology Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
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ABSTRACT |
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We have used isolated rat liver Golgi membranes to reconstitute the synthesis of sulfated glycosaminoglycans (GAGs) onto the membrane-permeable, external acceptor xyloside. Biosynthetic labeling of GAGs with [35S]sulfate in vitro is shown to have an absolute requirement for ATP and cytosolic proteins and is inhibited by dismantling the Golgi apparatus with okadaic acid or under mitotic conditions suggesting that inter-compartmental transport between Golgi cisternae is a prerequisite for the successful completion of the initiation, polymerization, and sulfation of GAGs. Accordingly, we show that in vitro synthesis of 35S-GAGs utilizes the same machinery employed in Golgi transport events in terms of vesicle budding (ADP-ribosylation factor and coatomer), docking (Rabs), targeting (SNAREs), and fusion (N-ethylmaleimide-sensitive factor). This provides compelling evidence that GAGs synthesis is linked to Golgi membrane traffic and suggests that it can be used as a complementation-independent method to study membrane transport in Golgi preparations from any source. We have applied this system to show that intra-Golgi traffic requires the function of the Golgi target-SNARE, syntaxin 5.
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INTRODUCTION |
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Glycosaminoglycans (GAGs)1 are sulfated polysaccharides that share a common pattern of structure consisting of an unbranched, alternating sequence of hexosamine (GlcN, GlcNAc, or GalNAc) and hexuronic acid (GlcUA or IdceA).
The biosynthesis of GAG chains involves the ordered, stepwise addition of a series of carbohydrate units onto the polypeptide backbone of a proteoglycan (for reviews see Refs. 1-3). The process starts with the addition of xylose (Xyl) to a Ser or Thr residue of the protein backbone. Two Gal residues and a GlcUA unit are then added sequentially to form the tetrasaccharide core linkage region (GlcUA-Gal-Gal-Xyl) common to most GAGs. Chain polymerization then starts with the addition of GlcNAc (in heparin/heparan sulfate) or GalNAc (in chondroitin sulfate and dermatan sulfate) residues alternating with GlcUA to form a chain containing up to 100 or more monosaccharide units. Polymerized sugars are then subjected to a series of modifications including N-deacetylation/N-sulfation, O-sulfation, and epimerization of GlcUA to IdceA.
It is important to note that many of these substitutions do not occur at random but only take place in a coordinated fashion after a previous modification has rendered the appropriate substrate available to the next modifying enzyme(s). Thus, the enzyme adding the second Gal in the linkage region (Gal transferase II) will only do so after the product of Gal transferase I (Xyl-Gal) is formed. N-Deacetylation (in heparan sulfate) is necessary for N-sulfation to occur, and N-sulfation is an absolute prerequisite for C5 epimerization of GlcUA to IdceA and for O-sulfation (4). N-Sulfation of the entire chain is terminated before O-sulfation is initiated (5). The biosynthetic process is fast; complete synthesis of an entire GAG chain is achieved in 1-3 min (6).
All the transporters necessary to translocate the sugar precursors (UDP-monosaccharide) and the sulfate donor (adenosine-3'-phospho-5'-phosphosulfate, PAPS) used as building blocks for GAGs are located on Golgi membranes (7). With the possible exception of Xyl addition to the polypeptide backbone, which probably occurs in the endoplasmic reticulum (ER), the biochemical machinery for the synthesis of the tetrasaccharide linkage region and for the chain polymerization, epimerization, and sulfation of GAGs is located inside the lumen of the Golgi apparatus (2, 8, 9).
Several lines of evidence suggest that the biochemical reactions for the synthesis of sulfated GAGs take place in separated membrane compartments of the Golgi complex and that, therefore, the GAG precursors attached to the backbone protein must be transported from one cisterna to the next one along the secretory pathway to undergo the ensuing biosynthetic reactions. Thus the first Gal addition by Gal transferase I to Xyl-Ser is a very early, probably cis-Golgi, modification, whereas addition of the second core Gal (by Gal transferase II) and GlcUA occur in a more distal compartment (at least medial-Golgi), as do the polymerization reactions, whereas sulfation is primarily a trans-Golgi modification. Evidence for this model comes from experiments in which intracellular membrane traffic is stopped either by the absence of cytosol or by the inclusion of transport inhibitors such as N-ethylmaleimide (NEM) or ilimaquinone; under these conditions GAG synthesis does not take place and only Gal-Xyl is made, with very little production of Gal-Gal-Xyl (10). This suggests that Gal transferases I and II are largely located in separate membrane compartments, a conclusion in good agreement with subfractionation studies in which both enzymes were shown to have a different density distribution (11). Another standard procedure to inhibit intracellular transport, treatment with brefeldin A (BFA), has been shown to block GAG synthesis in many systems (12-17). Even sulfation of fully formed, pre-loaded GAGs is inhibited by BFA (12, 18) despite the fact that this drug does not significantly affect the activity of the sulfotransferase (14). Indeed, GAGs can be sulfated in the presence of BFA if they are able to reach the TGN as is the case of proteoglycans recycling from the cell surface, as has been elegantly showed by Fransson et al. (19). These data are in agreement with the widespread idea that sulfate addition is a late Golgi modification. Consistent with this view is the fact that sulfated GAGs have been identified by cytochemical (20, 21) and immunoelectron microscopy techniques (22) in the medial- and trans-, but not the cis-, cisternae of the Golgi complex.
The requirement for a Xyl-polypeptide as the starting block to build a
GAG chain can be bypassed easily by the introduction of a mildly
hydrophobic Xyl analogue,
4-methylumbelliferyl--D-xyloside (xyloside). This
compound will cross the membranes and act as an acceptor for the
biosynthetic machinery involved in GAGs synthesis (23), thereby
competing with the endogenous acceptors (proteoglycans) by providing a
large number of nuclei onto which GAG synthesis can start. After
promoting initiation, polymerization, and sulfation onto xyloside, free
GAG chains are formed. These large molecules are highly acidic and
cannot cross the membranes back to the cytosol. They are transported
through the Golgi apparatus, probably in vesicular carriers, packaged
at the TGN into secretory vesicles to be transported to the cell
surface and finally released, after fusion with the plasma membrane,
into the extracellular milieu. Xyloside has been used to generate free
GAG chains in living cells for many years (24-26), and secretion of
xyloside-induced, sulfated GAGs is a well established marker of
constitutive secretion (27).
As virtually all cells tested are capable of synthesizing GAGs onto xyloside, an assay in which intra-Golgi traffic is measured, following synthesis of xyloside-attached GAGs, would provide a simple test to assess transport ability, should have universal application, and could be utilized to standardize Golgi transport characteristics and requirements across a wide range of systems. We decided, therefore, to attempt to establish such a system using isolated rat liver Golgi and xyloside.
We describe here the development of such an assay and show that sulfation of newly synthesized GAGs onto externally added xyloside is indeed coupled to intra-Golgi transport. Our evidence demonstrates that this system closely follows the characteristics of the in vivo process in terms of transport conditions, fusion, and coat proteins required. Additionally we show that the Golgi t-SNARE syntaxin 5 is involved in this process.
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EXPERIMENTAL PROCEDURES |
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Chemicals--
Chondroitinase AC was purchased from Oxford
Glycosystems; staurosporine and okadaic acid were from Calbiochem;
[35S]PAPS (2.5 Ci/mmol) and
Na235SO4 (carrier-free) were from
NEN Life Science Products; and GTPS was from Boehringer Mannheim.
All other chemicals were from Sigma.
Purification of Rat Liver Golgi Membranes-- Rat liver Golgi was purified as described (28). The final fraction was routinely checked by electron microscopy and found to be composed of a substantial proportion of stacked cisternae. Typical purification factors were 60-100-fold.
Collection of Interphase and Mitotic Cytosols from HeLa
Cells--
Interphase and mitotic cytosols were obtained from spinner
HeLa cells grown in the presence or absence or 0.1 µg/ml nocodazole as described (29). All cytosols were desalted before use with the aid
of a Bio-Rad P6-DG column. Interphase cytosols were desalted in 25 mM Hepes, pH 7.0, 25 mM KCl, 2.5 mM
MgCl2 (HKM buffer) containing 2 mM ATP. In
order to preserve the high protein kinase activity of the mitotic
cytosols, these were desalted in the same buffer containing the
following phosphatase inhibitors: 1.5 mM EGTA and 2 mM -glycerophosphate. Typical protein concentration of
cytosols was 10-15 mg/ml.
Cell Culture and Labeling with
[35S]Sulfate--
Mitotic HeLa cells obtained as above
were used for the estimation of the 35S-GAGs synthesizing
capacity. After the 24 h nocodazole treatment (resulting in 95%
mitotic cells), 5 × 105 cells were allowed to attach
for 2 h to 6-well plastic dishes coated with polylysine. The
medium was then removed, and the cells were washed three times with PBS
and incubated in buffer A (110 mM NaCl, 5.4 mM
KCl, 0.9 mM Na2HPO4, 10 mM MgCl2, 2 mM CaCl2, 1 g/liter glucose, 20 mM Hepes, pH 7.2) containing 0.5 mM xyloside as well as 0.1 µg/ml nocodazole for 20 min at
37 °C after which the medium was removed and 0.5 ml of
[35S]sulfate (0.25 mCi) in buffer A was added. After 10 min of labeling at 37 °C the medium was removed, and the cells were
washed five times with 20 mM unlabeled sulfate in PBS.
Cells were then extracted and processed for 35S-GAGs
determination by the cetylpyridinium chloride (CPC) precipitation technique as described (27).
Standard in Vitro Assay for 35S-GAGs
Synthesis--
A typical reaction contained, in 50 µl, 10 µg of
rat liver Golgi, 0.1 mg (protein) of HeLa cell interphase cytosol, 0.2 M sucrose, 25 mM Hepes, pH 7.0, 25 mM KCl, 2.5 mM MgCl2, 1 mM MnCl2, 1 mM DTT, a mixture of
UDP-sugars containing 0.5 mM each of UDP-Gal, UDP-GlcNAc,
and UDP-GlcUA, an ATP-regeneration system consisting of 1 mM ATP, 5 mM creatine phosphate, and 7 units of
creatine kinase, 0.5 mM xyloside, and 0.5 µCi of
[35S]PAPS. This mixture was incubated for 30 min at
37 °C in borosilicate glass tubes. The reaction was stopped by
addition of 100 µl of 7% trichloroacetic acid (final concentration).
After 30 min on ice the samples were transferred to 1.5 ml
microcentrifuge tubes and spun at 27,000 × g for 20 min. The supernatant was recovered and added to microcentrifuge tubes
containing 1 ml of 0.25 M sodium acetate in methanol and
kept at 20 °C or less for at least 1 h. After centrifugation
at 27,000 × g for 30 min, the supernatant was
discarded and the pellet air-dried and resuspended in 20 µl of
electrophoresis sample buffer. 15 µl were then loaded onto 15%
SDS-PAGE minigels and run at 300 V for about 1 h. The gels were
then briefly stained and de-stained and dried on paper. Dry gels were
exposed in a PhosphorImager cassette for at least 1 day after which the
accumulated radioactivity was quantified with the aid of a
PhosphorImager scanner. We used this procedure for determination of
35S-GAGs in our assay because the CPC precipitation
technique (which measures the radioactivity present in the precipitate
by scintillation counting) produced a very low signal in the in
vitro assay despite working very well with living cells.
Recombinant Proteins--
Bacterially expressed,
His6-tagged NSF and -SNAP were purified by nickel
affinity chromatography as described previously (31, 32). Recombinant
myristoylated ARF1 (33) and p47 (34) were prepared as described. For
the preparation of recombinant, soluble syntaxin 5, the cDNA coding
for the p35 form (see Ref. 35) minus the C-terminal transmembrane
domain (syntaxin 5-
TM) was amplified by polymerase chain reaction
and the fragment obtained cloned into the pGEX4T-2 vector. GST-syntaxin
5-
TM was then purified from extracts of transfected bacteria using
glutathione-Sepharose (Amerhsam Pharmacia Biotech), and the glutathione
S-transferase moiety was removed with thrombin to make
syntaxin 5-
TM as described (36). Syntaxin 5-
TM is hereby referred
to as soluble syntaxin 5. A His6-tagged version of mutant
Sar1 (H79G, GTP-restricted) was obtained as previously reported (37).
His6-tagged Rab-GDI was prepared according to Ullrich
et al. (38).
Purification of p97 and Coatomer-- Rat p97 was purified by anion exchange chromatography and density centrifugation as described (32). Coatomer was purified from rat liver cytosol as described by Waters et al. (39).
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RESULTS |
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In Vitro Synthesis of GAGs by Rat Liver Golgi-- Incubation of purified rat liver Golgi with xyloside, [35S]PAPS, and UDP-sugars under conditions that support vesicular transport (presence of cytosol and an ATP-regenerating system) resulted in the appearance of several bands corresponding to sulfated proteins as well as 35S-labeled material that ran as a smear in the low molecular weight area of SDS-polyacrylamide gels. More than 97% of the 35S-labeled products were precipitated by treatment with 7% trichloroacetic acid. The low molecular weight labeled material was, however, soluble in 7% trichloroacetic acid, thereby providing a convenient way of separating it from labeled proteins.
The soluble 35S-labeled products remaining in the trichloroacetic acid supernatant could be precipitated with 0.25 M sodium acetate in methanol. The pellet from this precipitation was solubilized in sample buffer and run on an SDS-polyacrylamide gel (Fig. 1A). A small peak of high molecular weight radioactive material was found in samples with or without xyloside (Rf
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Intra-Golgi Transport Is Required for Synthesis of Xyloside-attached Sulfated GAGs-- In order to demonstrate that the biosynthesis of 35S-GAGs is linked to membrane (presumably vesicular) transport, we first ruled out the possibility of synthesis by glycosylation of material that had diffused out of the lumen. Thus, membrane permeabilization with 0.05% Triton X-100 during the reaction abolished synthesis of sulfated GAGs (Fig. 2A) suggesting that properly sealed vesicles and cisternae are needed for efficient synthesis as would be expected from a process driven by vesicular transport.
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NSF, Rabs, and COPI but Not COP-II Components Are Used in GAGs
Transport--
The N-ethylmaleimide-sensitive factor (NSF)
is a protein first isolated as an NEM-inactivated component required at
the fusion stage of intra-Golgi transport (61, 62) and later shown to be crucial, by biochemical and genetic criteria, for many steps of
inter-compartmental transport (63, 64). To address the involvement of
this factor in our in vitro GAG assay, the reaction mixture
(both membranes and cytosol) was treated with 1 mM NEM on
ice for 15 min, after which the remaining reagent was quenched by
addition of 3 mM DTT. This manipulation inhibited
xyloside-induced 35S-GAG synthesis. However, the effect
could not by reversed by addition of fresh cytosol or purified
recombinant NSF (data not shown). The irreversibly inactivated process
lies in the Golgi membranes and not in the cytosol. It is likely that
this is the N-deacetylation of heparan sulfate, a
modification sensitive to 1 mM NEM (65) and an activity
absolutely required for subsequent sulfation of the GAG chain (4).
Treatment of cytosol alone with 1 mM NEM inactivated by
60-70% the capacity of isolated Golgi membranes to synthesize
35S-GAGs (Fig. 5). The
activity was restored by addition of 0.5 µg of purified recombinant
NSF (added together with 1.5 µg of -SNAP). Addition of NSF plus
-SNAP to intact cytosol had no effect (not shown) suggesting that
normal levels of cytosolic NSF are near-saturating. Only a slight
recovery could be achieved by addition of purified rat p97 (Fig. 5),
another NEM-sensitive protein believed to be involved in some
homotypic, rather than heterotypic, membrane fusion events (32, 66,
67). Our purified p97 was depleted in p47, a protein believed to be an
obligatory partner in p97 function (34). Nevertheless, even when we
supplemented the assay with recombinant p47 (up to 0.5 µg/assay)
alone or combined with p97, no increase in the signal was obtained (not
shown). Inclusion of a monoclonal antibody against NSF (2E5) decreased by 50% the 35S-GAG signal, and the inhibition was overcome
by the presence of 0.5 µg of NSF (Fig. 5). Comparable levels of
inhibition of intra-Golgi transport had been obtained with this
antibody in the in vitro transport assay developed by
Rothman and co-workers (68). This antibody has also been found to
inhibit transport (about 50%) from the ER to the plasma membrane
(69).
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Golgi SNAREs Participate in Intra-Golgi Transport of GAGs-- The current model to explain the specificity of vesicular transport (vesicles containing the right cargo fusing with the appropriate membrane) holds that vesicles will recognize their target membrane at the level of docking by controlling the pairing of cognate proteins present on the membranes that are to fuse. This idea arose from the identification of a family of proteins, present in vesicle and target membranes, capable of recognizing each other and able to bind crucial components of the fusion machinery, SNAPs (soluble NSF-attachment proteins). These proteins have been termed SNAREs (SNAP receptors) (85). Vesicle and target membrane SNAREs (v- and t-SNAREs) have been identified in different intracellular compartments. There is very strong evidence implicating SNAREs in many steps of membrane traffic (for reviews see Refs. 80, 86, and 87).
Several t- and v-SNAREs have been localized in the Golgi apparatus so far. It was obviously interesting to know whether these proteins would be involved in intra-Golgi transport, and therefore, we tried to address this question using the in vitro Golgi transport assay described in this study. The best known of Golgi SNAREs is syntaxin 5 (homologue of yeast Sed5), a t-SNARE localized mainly to the cis side of the Golgi complex (35, 88, 89) where it has been shown to be required for the forward transport of vesicles arising from the ER (88). Inclusion in our reaction mixture of 1 µg of an affinity purified polyclonal antibody raised against recombinant rat syntaxin 5 (35) potently inhibited synthesis of 35S-GAGs (Fig. 7A). A monoclonal antibody against a Golgi v-SNARE (GOS-28) also blocked GAG synthesis (i.e. transport) (Fig. 7A). GOS-28 had been previously shown to be required in ER to Golgi and cis- to medial-Golgi transport (90, but see Ref. 91). The block in intra-Golgi transport caused by the anti-syntaxin 5 antibody was unexpected as syntaxin 5 had been reported not to be involved in intra-Golgi transport (88). To substantiate this finding we prepared recombinant soluble syntaxin 5 and added it in increasing amounts to our reaction mixture. The prediction was that a true requirement of syntaxin 5 for Golgi transport would mean inhibition of the reaction after flooding the system with a SNARE unattached to membranes to compete with the endogenous, membrane-attached protein for the pairing with v-SNAREs. This is indeed what we found (Fig. 7B). Submicrogram amounts of purified recombinant syntaxin 5 inhibited the synthesis of GAGs in a dose-dependent manner. In addition, low amounts of the recombinant protein reversed the inhibition brought about by the anti-syntaxin 5 antibody (Fig. 7C), stressing the specificity of the blocking.
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DISCUSSION |
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We present several lines of evidence that point to vesicular transport as being essential for GAG synthesis. First, there is an absolute requirement for transport conditions (ATP, cytosol, and physiological temperature); second, fragmentation of the Golgi apparatus during mitosis or under okadaic acid treatment (procedures known to arrest membrane transport) brings about a blocking of GAG biosynthesis; third, the process clearly requires the function of proteins involved in promoting membrane fusion (NSF) and in regulating docking (Rabs); fourth, synthesis utilizes proteins implicated in COP-I vesicle budding (ARF and coatomer); fifth, v- and t-SNAREs (GOS-28 and syntaxin 5), key components of the vesicle targeting machinery, are employed during the biosynthetic process.
This study reports the generation of sulfated GAGs onto an external acceptor in a cell-free system. In order to achieve this we had to take particular note of the specific requirements for membrane transport, especially the presence of cytosol and ATP. It is at present unclear how many unitary transport steps are necessary to complete a single, sulfated xyloside-attached GAG chain because the exact localization of the biosynthetic reactions remains to be described and can only be, with the available data, tentatively allocated. The starting reaction (addition of the first Gal residue to xyloside) is believed to be an early Golgi event (88) although this could be the cis- or the medial-Golgi, and it is certainly separated from the next biosynthetic step (addition of the second Gal to Gal-Xyl) as shown in transport-arrested systems (10). The site of polymerization reactions is not clear-cut. Kinetic studies suggest it precedes sulfation (92, 93) although there is some evidence that sulfated GAGs can be further polymerized (94). However, sulfated GAGs are not formed if vesicular transport is stopped (this study and Refs. 12 and 18). The finding that GAG sulfation appears to be an all or nothing process, with some chains fully sulfated and others not sulfated at all (93), could be easily explained if there were separate compartments for polymerization and sulfation. Given the fact that GAG sulfation appears to be a trans-Golgi/TGN event (2, 18, 95), this would leave the polymerization reaction in the medial/trans compartment of the Golgi apparatus. Thus the completion of a GAG chain may be tentatively divided as follows: initiation, cis/medial-Golgi; polymerization, medial/trans-Golgi; and sulfation, trans/TGN. According to this view our in vitro system of GAG synthesis would require at least two inter-compartmental transport events. Nevertheless, the biosynthesis of a complete, sulfated GAG chain involving only one inter-compartmental transport step (separating, for instance, initiation from polymerization + sulfation) would still be compatible with the data presented in this study. Both the evidence we provide and that available in the literature cannot rule out this possibility.
The only cell-free intra-Golgi transport assay available until now is the well known system set up by Rothman and co-workers (96, 97) in which transport of VSV-G between cis- and medial-Golgi is measured. This assay has been extremely useful in unravelling the components involved in membrane transport. It requires, however, a mutant CHO cell line as well as virus-infected cells. Besides, only transport to the medial-Golgi is measured with the result that components specifically required for transport from the medial to the trans compartment would remain overlooked. An early report describing transport of VSV-G to the trans-Golgi in this cell-free system by measuring sialylation of VSV-G (98) has only rarely been used. It seems that biosynthetic transport of proteins from the ER to the trans/TGN can only be reliably reconstituted in permeabilized cells. Attempts to detect sialylation of VSV-G with perforated CHO cells were unsuccessful (99), but the system works well with perforated normal rat kidney cells (56). Nonetheless, identification of a component required for the last step in intra-Golgi transport would be difficult to achieve using the semi-intact cells approach if that component is also involved in ER to Golgi transport. The cell-free GAG assay we have developed in the present study should work with Golgi membranes isolated from other sources. This assay is absolutely linked to sulfation, a modification occurring in the most distal Golgi compartment. It might, therefore, allow the investigation and identification of components required for transport to the trans/TGN compartment without the need for a previous obligatory ER to Golgi step indispensable in the protein transport systems using semi-intact cells.
The most surprising result of this study is our finding that syntaxin 5 is required for GAGs synthesis (i.e. intra-Golgi transport). Syntaxin 5 is a t-SNARE located in the cis-Golgi area that has been shown to be required for ER to Golgi traffic (88). As expected, it has been isolated in association with several v-SNAREs from the ER (100). From this evidence it would appear that syntaxin 5 is the target molecule recognized by incoming ER vesicles, an interpretation in line with the original formulation of the SNARE hypothesis (85). However, Sed5, the yeast analogue of mammalian syntaxin 5, functionally interacts with v-SNAREs from other compartments such as Golgi Sft1 (101) and Vti1p from the vacuole (102) indicating its participation in membrane transport events further along the secretory pathway. Some Sed5 mutants can, under certain conditions, accumulate invertase (a secretory protein) in an early Golgi compartment indicating that Sed5 might be required for some transport step to the distal Golgi (101). In agreement with this view, our finding of syntaxin 5 involvement in 35S-GAGs synthesis could be explained if syntaxin 5 is required for medial- to trans/TGN traffic as blocking of this step would preclude sulfation. This effect could arise directly from a position of syntaxin 5 as a t-SNARE in a trans-Golgi compartment. Although the bulk of syntaxin 5 has been localized to the cis area of the Golgi complex, this does not rule out the presence of small amounts of syntaxin 5 in the trans/TGN side. Alternatively, syntaxin 5 in the cis-Golgi could be required for the arrival of recycling vesicles from distal Golgi compartments and would only be indirectly required in forward intra-Golgi transport. It is well established that mutants in which retrograde membrane traffic is defective are also affected in forward transport (103, 104) presumably because of the need to recycle essentials components for anterograde movement. Both possibilities could explain our finding about the inactivation of GAG biosynthesis when syntaxin 5 function is impaired.
The in vitro GAG synthesis assay described in this paper provides an alternative system to follow intra-Golgi transport and should be useful to detect previously overlooked components of this pathway such as syntaxin 5.
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ACKNOWLEDGEMENTS |
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We thank Regina Kieckbush for excellent
technical help. We are grateful to V. N. Subramanian and Wanjing
Hong for the gift of anti-GOS-28 and anti-mSec13 antibodies; Mitsuo
Tagaya for the anti-NSF monoclonal antibody; Wieland Huttner for a
batch of [35S]PAPS; David Shima for the preparation of
recombinant mutant Sar1; Francis Barr for recombinant Rab-GDI and
purified coatomer; Norman Hui for the preparation of anti-syntaxin 5 antibodies; Hisao Kondo for the gift of purified p97, recombinant p47,
and syntaxin 5 (p35); Tim Levine and Joyce Muller for the preparation recombinant -SNAP and NSF; Felicia Hunte and Francis Barr for recombinant myristoylated ARF; and all the members of the Warren lab
for many helpful discussions. We also thank Marino Zerial, Richard
Kahn, Wally Whiteheart, Bill Balch, and Jim Rothman for the kind gift
of the cDNAs used to produce the recombinant proteins employed in
this study.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Postdoctoral fellow of the European Union (Biotechnology
program).
§ To whom correspondence should be addressed. Tel.: 171-269-3561; Fax: 171-269-3412; E-mail: g.warren{at}icrf.icnet.uk.
1
The abbreviations used are: GAG,
glycosaminoglycan; ARF, ADP-ribosylation factor; BFA, brefeldin A; CHO,
Chinese hamster ovary; CPC cetylpyridinium chloride; DTT,
dithiothreitol; ER, endoplasmic reticulum; GDI, guanine nucleotide
dissociation inhibitor; GTPS, guanosine-5'-O-[3-thiotriphosphate]; IdceA,
L-iduronic acid; NEM, N-ethylmaleimide; NSF,
N-ethylmaleimide-sensitive factor; PAPS, adenosine-3'-phospho-5'-phosphosulfate; PBS, phosphate-buffered saline;
SNAPs, soluble NSF attachment proteins; SNAREs, soluble NSF attachment
protein receptors; v- and t-SNAREs, vesicle and target membrane SNAREs;
TGN, trans-Golgi network; VSV-G, vesicular stomatitis virus
G protein; xyloside, 4-methylumbelliferyl-
-D-xyloside.
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REFERENCES |
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