Functional and physical interactions of the adaptor protein complex AP-4 with ADP-ribosylation factors (ARFs)

AP-4 is a member of the family of heterotetrameric adaptor protein (AP) complexes that mediate the sorting of integral membrane proteins in post-Golgi compartments. This complex consists of four subunits (epsilon, beta4, mu4 and sigma4) and localizes to the cytoplasmic face of the trans-Golgi network (TGN). Here, we show that the recruitment of endogenous AP-4 to the TGN in vivo is regulated by the small GTP-binding protein ARF1. In addition, we demonstrate a direct interaction of the epsilon and mu4 subunits of AP-4 with ARF1. epsilon binds only to ARF1-GTP and requires residues in the switch I and switch II regions of ARF1. In contrast, mu4 binds equally well to the GTP- and GDP-bound forms of ARF1 and is less dependent on switch I and switch II residues. These observations establish AP-4 as an ARF1 effector and suggest a novel mode of interaction between ARF1 and an AP complex involving both constitutive and regulated interactions.     

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.     

Interspecies relationships among ADP-ribosylation factors (ARFs): Evidence of evolutionary pressure to maintain individual identities

ADP-ribosylation factors (ARFs) are ～20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.     

ADP-ribosylation factors (ARFs) and ARF-like 1 (ARL1) have both specific and shared effectors: characterizing ARL1-binding proteins

Despite the 40-60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1. Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously.     

ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.     

CiS- and trans-acting factors involved in centromere function in Saccharomyces cerevisiae

The function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region 78 to 86bp in length which is greater than 90% A + T, usually in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8bp sequence and on the other side by a highly conserved 25bp sequence which contains partial dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of each subset of the consensus sequence to centromere function. A protein which binds to the 8bp sequence and at least one that binds to the 25bp sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation.     

ADP-ribosylation factor 6 (ARF6) defines two insulin-regulated secretory pathways in adipocytes.

ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.     

Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion.

We have used an in vitro endosome fusion assay, recombinant ARF, synthetic peptides, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to study the role of ARF during endocytosis. Previous work has shown that GTP gamma S stimulates in vitro endosome fusion in dilute cytosol (less than 0.5 mg/ml) but inhibits fusion in concentrated cytosol (greater than 1.0 mg/ml). Two peptides corresponding to the NH2-terminal 16 amino acids of human ARF1 and ARF4 blocked GTP gamma S stimulation of fusion in dilute cytosol and reversed GTP gamma S inhibition of fusion in concentrated cytosol. The addition of recombinant human ARF1 to endosomes in dilute or concentrated cytosol resulted in GTP gamma S-dependent inhibition of fusion. Only the myristoylated form of ARF inhibited fusion. The NH2-terminal ARF1 peptide reversed inhibition by recombinant ARF1. Preincubation experiments showed that endosomes could form an ARF-resistant intermediate during the fusion process. Western blot analysis revealed clathrin-coated vesicles extracted with detergent retained ARF. The results suggest that ARF is involved in both the stimulatory and inhibitory effects of GTP gamma S in dilute and concentrated cytosol, respectively. Furthermore, myristoylation, the NH2-terminal domain, and binding to GTP appear to be critical for ARF activity during an early prefusion step required for endocytosis.     

Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.     

Identification and analysis of a DNA fragment from Saccharomyces kluyveri that can complement the loss of CDC25 function in Saccharomyces cerevisiae.

In the budding yeast, Saccharomyces cerevisiae, the function of wild-type Ras proteins is dependent on the CDC25 protein, which promotes the exchange of guanine nucleotides bound to Ras. To facilitate the identification of proteins which similarly regulate Ras function in higher eukaryotes, we have identified the CDC25 gene from another budding yeast, Saccharomyces kluyveri, by low-stringency hybridization to an S. cerevisiae CDC25 restriction fragment. This protein, SKCDC25, shares significant amino acid homology with CDC25, SCD25, and Ste6 of Schizosaccharomyces pombe in the C-terminal portion of the protein. The expression of SKCDC25 in a temperature-sensitive cdc25 strain of S. cerevisiae complements the loss of endogenous CDC25 activity. The identification of the highly conserved C-terminal sequences, which direct bona fide CDC25 activity within these proteins, will aid in the isolation of CDC25 genes from higher eukaryotes.     

Nystatin effects on vacuolar function in Saccharomyces cerevisiae.

The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ～200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.     

Sterol 24(28) methylene reductase in Saccharomyces cerevisiae.

Optimal conditions for the 24(28)methylene reductase were obtained. The enzyme assay provided for unusually high activity; the Km was determined to be 10.8 mum. The enzyme activity was increased in cells grown with ethanol as the substrate.     

Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.     

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.