On the action of Brefeldin A on Sec7-stimulated membrane-recruitment and GDP/GTP exchange of Arf proteins.

Arf (ADP-ribosylation factor) proteins form a special class of small GTP-binding proteins in that their activation by GDP/GTP exchange is coupled to their recruitment to membranes using a built-in structural mechanism. These coupled processes are stimulated by GEFs (guanine nucleotide-exchange factors) that carry a catalytic Sec7 domain, whose basic mechanism has been uncovered by biochemical and structural studies. Crystal structures of intermediates of the GDP/GTP exchange reaction, from which GDP has not dissociated, notably allowed a movie of the exchange reaction to be reconstituted. They showed that Sec7 domains secure Arf-GDP to membranes before they proceed to nucleotide dissociation, and thus are active participants to the coupling of membrane-recruitment to nucleotide exchange. The drug BFA (Brefeldin A) was used to trap the complex that initiates the exchange reaction, providing a structural basis for its inhibition of Arf and its action on the membrane-recruitment of isolated Sec7 domains. Based on the dissection of this basic mechanism, the survey of reported BFA effects in cells on large multidomain ArfGEFs of the BIG1/2 and GBF1 families shows that the levels and compartmental distribution of BFA-induced recruitment of ArfGEFs to membranes cannot be explained from isolated Sec7 domains acting as independent domains. This leads to the hypothesis that Sec7 activity is inhibited in these ArfGEFs by an intramolecular interaction, which would be released by interaction with a compartment-specific receptor.

Inhibition of CMP-sialic acid transport into Golgi vesicles by nucleoside monophosphates.

We examined the interactions of nucleotides with the CMP-sialic acid transporter in order to better understand which features play a role in binding and to investigate the relationship between binding and subsequent transport. With respect to the sugar, the transporter requires a complete ribose ring for tight binding, and the 2'-ara hydrogen makes an important contact. The enzyme exhibits little specificity with respect to the 2'- and 3'-hydroxyls, as it tolerated substitutions ranging from fluorine to an azido group. In the base, the C4 amine and C2 carbonyl groups make important contacts, while the N3 nitrogen does not. However, adding a methyl group to N3 dramatically reduced binding, indicating that mass at this position sterically hinders binding. Adding a group at C5 had either no effect or slightly enhanced binding. To determine if the transporter recognizes these CMP analogues as substrates, we assayed them for their ability to trans stimulate CMP-sialic acid import. These data suggest that the enzyme transports a wide variety of NMPs, and the rate of transport is inversely proportional to the K(I) of the analogue. The importance of our findings for understanding the specificities of the different nucleotide-sugar tranlocators and the design of novel glycosylation inhibitors are discussed.

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells.

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melançon, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melançon, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.

3'-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides.

In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.

Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.

p200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A.

The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism. Here, we investigated the cellular localization and BFA sensitivity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53/58. Analysis of several truncated forms mapped the Golgi-localization signal to the N-terminal third of p200. BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5'-[gamma-thio]triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 microM. On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disruption of Golgi morphology. p200 mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.

GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

Fusogenic domains of golgi membranes are sequestered into specialized regions of the stack that can be released by mechanical fragmentation.

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.

Human GBF1 is a ubiquitously expressed gene of the sec7 domain family mapping to 10q24.

We present the cDNA sequence of human GBF1 along with data on expression pattern and genomic attributes. The deduced polypeptide encodes a putative guanine nucleotide exchange factor of 206.5 kDa, containing a centrally positioned Sec7 domain and a proline-rich region at the extreme C terminus. Its mRNA transcripts are found in 17 tissues and cell lines, likely suggesting a housekeeping role for GBF1p. The gene maps to 10q24, 2.33 cR from D10S540. It is fully contained within YAC 822C1 and covers at most 450 kb. Its Sec7 domain-encoding region harbors four introns.

Analysis of recombinant human ADP-ribosylation factors by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry.

Two complementary approaches utilizing reverse-phase high-performance liquid chromatography and liquid chromatography/mass spectrometry were developed to analyze recombinantly produced Group I and Group II human ADP-ribosylation factors (ARFs). We observe that the NH2 termini of Group II ARFs (ARF4 and ARF5) are efficiently processed by removal of the initiating methionine. In contrast, the NH2 termini of Group I ARFs (ARF1 and ARF3), although fully deformylated, undergo only partial methionine cleavage. This result is unexpected as ARFs are canonical substrates for methionine processing in both bacterial and eukaryotic systems, but it may explain the difficulties encountered by many researchers attempting to produce myristoylated ARFs in Escherichia coli. Additionally, we observe that a significant fraction of purified ARF4 contains a modification which we demonstrate to be consistent with mono-glutathionation. Both methionine retention and glutathione modification may impact ARF function and the methods presented here should be employed to determine the quality of recombinant ARFs.

Human ARF4 expression rescues sec7 mutant yeast cells.

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.

Inhibition of UDP-N-acetylglucosamine import into Golgi membranes by nucleoside monophosphates.

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol.

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.

Mass-spectrometric analysis of ADP-ribosylation factors from bovine brain: identification and evidence for homogeneous acylation with the C14:0 fatty acid (myristate).

The two proteins from bovine brain previously shown to be required for the guanosine 5'-[gamma-thio]triphosphate-dependent inhibition of a well-characterized intra-Golgi transport assay, termed GGBF and GGBF, have been definitively identified as members of the ADP-ribosylation factor (ARF) family by electrospray MS analysis of the intact proteins, and of their tryptic fragments. Extensive protein-sequence information obtained from this analysis identified GGBF and GGBF as bovine ARF1 and ARF3 respectively. The sequence of bovine ARF3, which had not previously been determined, appears identical to that predicted from the rat and human ARF3 cDNAs. Further analysis of the N-terminal tryptic fragments of both bovine ARFs demonstrates N-terminal acylation solely with the C14:0 fatty acid (myristate). This finding establishes that the previously reported specific-activity difference between ARF1 and ARF3 in the intra-Golgi transport assay is not due to lipid heterogeneity at the N-terminus. This finding also indicates that the heterogeneity of N-terminal fatty-acyl groups previously observed on other myristoylated proteins is not universal.

3'-Azidothymidine (zidovudine) inhibits glycosylation and dramatically alters glycosphingolipid synthesis in whole cells at clinically relevant concentrations.

Recent in vitro work with Golgi-enriched membranes showed that 3'-azidothymidine-5'-monophosphate (AZTMP), the primary intracellular metabolite of 3'-azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al. (1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 microM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication.

Cytosolic ADP-ribosylation factors are not required for endosome-endosome fusion but are necessary for GTP gamma S inhibition of fusion.

A specific role for ADP-ribosylation factors (ARFs) in in vitro endosome-endosome fusion has been proposed (Lenhard, J. M., Kahn, R. A., and Stahl, P. D. (1992) J. Biol. Chem. 267, 13047-13052). However, in vivo studies have failed to support a function for ARFs in the endocytic pathway, since an antagonist of ARF activities, brefeldin A, does not interfere with receptor internalization (Schonhorn, J. E., and Wessling-Resnick, M. (1994) Mol. Cell. Biochem. 135, 159-164). This controversy surrounding the exact function of ARF in endocytic vesicle traffic prompted us to critically re-examine the involvement of ARFs in cell-free endosome fusion. Cytosol depleted of ARF activity was capable of supporting in vitro endocytic vesicle fusion but failed to support inhibition of this reaction in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). Addition of purified ARF1 restored the ability of the ARF-depleted cytosol to inhibit endosome fusion when incubated with GTP gamma S. Both endocytic vesicle fusion and the GTP gamma S-mediated inhibition of vesicle fusion were unaffected by brefeldin A. Moreover, the ATP requirement and kinetics of cell-free fusion are not altered by brefeldin A or depletion of cytosolic ARFs. These results suggest that cytosolic ARFs are not necessary for endosomal vesicle fusion in vitro but are responsible for inhibition of fusion in the presence of GTP gamma S and cytosolic factors in a brefeldin A-resistant manner.

Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A.

22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle-specific coats.

3'-Azido-3'-deoxythymidine potently inhibits protein glycosylation. A novel mechanism for AZT cytotoxicity.

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (HIV) infection. Unfortunately, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythymidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This inhibition results from direct competition with several pyrimidine-sugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve 3'-azido-3'-deoxythymidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects; thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.