2604 Research Article The Arf1p GTPase-activating Glo3p executes its regulatory function through a conserved repeat motif at its C-terminus

Natsuko Yahara1,*, Ken Sato1,2 and Akihiko Nakano1,3,‡ 1Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute and 2PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama 351-0198, Japan 3Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan *Present address: Department of Biochemistry, University of Geneva, Sciences II, Geneva, Switzerland ‡Author for correspondence (e-mail: [email protected])

Accepted 21 March 2006 Journal of Cell Science 119, 2604-2612 Published by The Company of Biologists 2006 doi:10.1242/jcs.02997

Summary ADP-ribosylation factors (Arfs), key regulators of ArfGAP, Gcs1p, we have shown that the non-catalytic C- intracellular membrane traffic, are known to exert multiple terminal region of Glo3p is required for the suppression of roles in vesicular transport. We previously isolated eight the growth defect in the ts mutants. Interestingly, temperature-sensitive (ts) mutants of the yeast ARF1 , Glo3p and its homologues from other harbor a which showed allele-specific defects in protein transport, well-conserved repeated ISSxxxFG sequence near the C- and classified them into three groups of intragenic terminus, which is not found in Gcs1p and its homologues. complementation. In this study, we show that the We name this region the Glo3 motif and present evidence overexpression of Glo3p, one of the GTPase-activating that the motif is required for the function of Glo3p in vivo. of Arf1p (ArfGAP), suppresses the ts growth of a particular group of the arf1 mutants (arf1-16 and arf1-17). Other ArfGAPs do not show such a suppression activity. Supplementary material available online at All these ArfGAPs show sequence similarity in the ArfGAP http://jcs.biologists.org/cgi/content/full/119/12/2604/DC1 catalytic domain, but are divergent in the rest of molecules. By domain swapping analysis of Glo3p and another Key words: ArfGAP, Glo3p, Glo3 motif Journal of Cell Science Introduction pairs of mutant alleles, both for cell growth and intracellular ADP-ribosylation factors (Arfs) constitute a ubiquitous family transport. These results demonstrated that the single Arf1 of small in eukaryotes, and are now recognized protein is indeed involved in many different steps of as essential components in membrane traffic. There are six intracellular transport in vivo (Yahara et al., 2001). However, Arf proteins in mammals and three Arfs in the yeast the mechanism of managing these various steps of transport by Saccharomyces cerevisiae, which are classified into three and Arf1p remained unsolved. two classes, respectively. These Arf proteins regulate formation Our next interest was to understand how the single Arf1p of vesicles in a variety of steps of membrane traffic by can fulfill such divergent functions. Many Arf interactors are interacting with coat components and also appear to control known to date, including guanine nucleotide exchange factors phospholipid and cytoskeletal organization in (GEFs), GTPase-activating proteins (GAPs) and effectors various ways (Donaldson and Jackson, 2000; Kirchhausen, (Donaldson et al., 2005; Nie et al., 2003). ArfGEFs and 2000; Nie et al., 2003; Schekman and Orci, 1996). Different ArfGAPs contain the Sec7 and the ArfGAP domains, classes of Arfs are thought to function at different places within respectively. In the , at least 15 ArfGEFs and a cell. Evidence is also present that a single Arf species may 16 ArfGAPs are present, and four ArfGEFs and four ArfGAPs have multiple roles (Donaldson et al., 2005; Donaldson and exist in S. cerevisiae, indicating the presence of complicated Jackson, 2000). In yeast, among the three Arf proteins, Arf1p networks for the regulation of Arfs. The yeast arf1 ts mutants and Arf2p are 96% identical in amino acid sequences. They we constructed may have different abilities to interact with execute redundant essential functions for growth and their such a variety of regulators and targets and thus express double knockout is lethal (Stearns et al., 1990), whereas Arf3p different downstream activities. is dispensable. For the purpose of identifying the molecules that execute the We previously isolated eight temperature-sensitive (ts) pleiotropic functions downstream Arf1p, our arf1 ts mutants mutants of the yeast ARF1 gene by PCR-based random are very useful because the multiple roles of Arf1p can be mutagenesis in the arf2⌬ background. These arf1 ts mutants dissected by different mutant alleles. One straightforward showed a variety of transport defects and morphological genetic approach is to screen for multicopy suppressors of alterations in an allele-specific manner. Furthermore, these arf1 ts mutants. The eight arf1 ts mutants we constructed intragenic complementation was observed between certain have been classified into three groups by intragenic Roles of the noncatalytic region of ArfGAPs 2605

complementation tests. arf1-16 and arf1-17 belong to the same group and show very similar phenotypes in protein transport. In both arf1-16 and arf1-17 alleles, glutamic acid at position 41 is mutated to valine (E41V), and arf1-16 has one additional mutation, D129E. These mutant cells have severe lesions in the Golgi-to-ER retrieval, not only by COPI- and Rer1p-dependent systems but also by the HDEL-receptor-mediated mechanism. They also show retardation of ER-to-Golgi transport of carboxypeptidase Y and secretion of underglycosylated invertase. Here, we report that the temperature sensitivity of arf1-16 and arf1-17 alleles is suppressed by the overexpression of Glo3p, but not by other ArfGAPs. We also describe the presence of a novel sequence motif in the C-terminal region of Glo3p, which is important for its suppression activity.

Results GLO3 is a multicopy suppressor of arf1-16 and arf1-17 To identify that participate in multiple functions of Arf1p, we screened the S. cerevisiae genomic DNA library on a multicopy vector for plasmids that suppress the growth defect of arf1-16 at 35°C. In addition to authentic ARF1 and ARF2, several clones containing different genomic loci were obtained. Subcloning and sequencing analysis identified five genes, GLO3, BET1, BOS1, OLE1 and GYP6, as suppressors of arf1- 16 (Fig. 1). Among them, GLO3, encoding an ArfGAP, showed the strongest suppression activity on arf1-16. Growth of the arf1-17 allele, which has the same E41V mutation and is classified in the same intragenic complementation group with arf1-16, was also rescued by GLO3 (Fig. 1).

Of the ArfGAPs, only GLO3 can suppress arf1-16 and arf1-17 In S. cerevisiae, at least four genes (GLO3, GCS1, AGE1 and AGE2) are reported to encode ArfGAP (Poon et al., 1999; Poon

Journal of Cell Science et al., 2001b; Poon et al., 1996; Zhang et al., 2003). In contrast to GLO3, however, overexpression of GCS1, AGE1 or AGE2 did not suppress the ts growth of arf1-16 and arf1-17 at all. In Fig. 1. Suppression activity of ArfGAPs, BET1, BOS1, OLE1, and the case of GCS1, its overexpression even exaggerated the GYP6 for arf1-16 and arf1-17 ts mutants. (A) arf1-16 (NYY16-1) growth defect of arf1-16 and arf1-17, but had no effect on the and arf1-17 (NYY17-1) cells were transformed with pNY24-GLO3 wild type (Fig. 1). The expression levels of GLO3 and GCS1 (2 ␮ GLO3), pNY24-GCS1 (2 ␮ GCS1), pNY24-AGE1 (2 ␮ AGE1), in arf1-16 and arf1-17 cells under these conditions were pNY24-AGE2 (2 ␮ AGE2), and the transformants were grown at confirmed by immunoblotting of HA-tagged proteins 23°C. Cells were diluted to a final concentration of 1.0 at OD 600, ␮ (supplementary material Fig. S1). The temperature sensitivity and 10 l of 10-fold dilutions were spotted on MCD (minimal of other arf1 ts mutants (arf1-11, -13, -14 and -18) was not glucose casamino acid medium) (–Trp) plates. The plates were incubated at the indicated temperatures for 2 days. Integrants of suppressed by the overexpression of any of these ArfGAPs wild-type ARF1 (WT, NYY0-1) and mutants transformed with (supplementary material Fig. S2), indicating that the pYO324 (vector) were used as controls. (B) arf1-16 and arf1-17 cells suppression was arf1-allele specific and GLO3 specific. were transformed with pNY25-BET1 (2 ␮ BET1), pNY25-BOS1 (2 ␮ BOS1), pNY25-OLE1 (2 ␮ OLE1), and pNY25-GYP6 (2 ␮ arf1-17p mutant protein shows a severe defect in GDP GYP6), and the transformants were spotted on MVD (–Leu) plates binding and grown as described in A. Integrants of wild-type ARF1 (WT) and Because the ts growth of arf1-16 and arf1-17 was suppressed mutants transformed with pYO325 (vector) were used as controls. by the overexpression of one ArfGAP, we examined nucleotide-binding activities of these arf1 mutant proteins. arf1-16 or arf1-17 protein was co-expressed with N- myristoyltransferase in E. coli, and purified by DEAE- procedure of wild-type (WT) Arf1p was not directly applicable Sepharose and gel-filtration chromatography as described in to arf1-16p. The tendency for oligomerization was much Materials and Methods. Some portions of arf1-16p and arf1- smaller in the case of arf1-17p; 60-70% of arf1-17p was eluted 17p behaved as a much larger protein perhaps due to self- in fractions of the same molecular size as WT-Arf1p (~20 oligomerization. In the case of arf1-16p, most of the protein kDa). So we decided to use arf1-17p for the biochemical was eluted in fractions corresponding to a molecular mass of analysis. Stoichiometric myristoylation was confirmed for both around 160 kDa (data not shown), therefore the purification WT-Arf1p and arf1-17p. 2606 Journal of Cell Science 119 (12)

The purified Arf1 proteins were then subjected to the in the GTP-bound active form, interacts with both Glo3p and analysis of guanine-nucleotide binding activities. Aliquots of Gcs1p in a two-hybrid system (Eugster et al., 2000). We Arf1p were mixed with [35S]GTP␥S or [3H]GDP and examined the interaction of arf1-16p/17p with these ArfGAPs incubated at 23 or 30°C in the presence of using the same two-hybrid system. We generated two-hybrid dimyristoylphosphatidylcholine (DMPC)/cholate micelles as constructs expressing N-truncated (⌬N17) versions of described previously (Kahn and Gilman, 1986). The resulting Arf1(Q71L)p, arf1-16(Q71L)p, or arf1-17(Q71L)p as fusions complex was trapped on a nitrocellulose filter and washed, to the LexA DNA-binding domain in the bait vector (pGlida), and then the guanine nucleotide bound to Arf1p was and then tested for interaction with ArfGAPs (Glo3p and quantified by scintillation counting. Fig. 2 shows the time Gcs1p) fused to the B42 activation domain in the prey vector course of GTP␥S- and GDP-binding to the WT and arf1-17 (pJG4-5) (Fig. 3). All chimeric proteins were expressed under proteins. For WT-Arf1p, the binding kinetics was almost the the GAL1 promoter and their expression levels were confirmed same at 23 and 30°C. By contrast, arf1-17p showed a clear by immunoblotting (supplementary material Fig. S3). We temperature sensitivity in nucleotide binding. The GDP could observe that both Glo3p and Gcs1p interact with binding of arf1-17p at 30°C plateaued during the first 90 Arf1(Q71L)p as described previously. By contrast, neither of minutes of incubation, the level of which was about twofold them showed positive interaction with arf1-16(Q71L)p or arf1- lower than the maximal value at 23°C. GTP␥S binding of 17(Q71L)p. arf1-17p was also lower at 30°C, but not as defective as GDP Next we tested the GAP activities of Glo3p and Gcs1p binding. These results suggest that arf1-17p has a less toward arf1-17p in vitro. Recombinant Glo3p and Gcs1p were defective binding to GTP than to GDP at 30°C. If the ts defect produced in E. coli and subjected to the in vitro GAP assay of arf1-17 cells is related to such imbalance of GTP/GDP using purified WT-Arf1p and arf1-17p as substrates (Fig. 4). binding at high temperature, it may be that this defect is Glo3p and Gcs1p stimulated the hydrolysis of GTP to GDP by suppressed by the overproduction of ArfGAP. WT-Arf1p at both 23 and 35°C. The GTPase activity of arf1- 17p was also enhanced by these ArfGAPs. The stimulatory Glo3p and Gcs1p show similar GAP activities toward effects were almost comparable to those of the wild type at arf1-17p in vitro 23°C, but were significantly lower at 35°C. However, what The above finding led us to speculate that different affinity of should be noted here is that the effects of Glo3p and Gcs1p on Glo3p and Gcs1p to arf1-16p/17p would explain their different the arf1-17 mutant protein were almost indistinguishable in effects on the ts growth of the mutants. We tested the this in vitro reaction. interaction between ArfGAPs and arf1-16p/17p using two methods, the two-hybrid analysis and the in vitro GAP assay. The C-terminal domain of Glo3p is important for its It has already been reported that an N-terminal-truncated form suppression activity on arf1-16 (⌬N17) of Arf1p with the Q71L mutation, which is stabilized How can we then explain the difference between Glo3p and

Journal of Cell Science 0.4

WT-Arf1p GDP 23˚C 0.5 arf1-17p

0.3 GDP 23˚C GDP 30˚C 0.4

0.3 0.2

0.2 GDP 30˚C 0.1 0.1 Bound nucleotide Bound (pmol) (pmol) / Arf1p 0 nucleotide Bound (pmol) / arf1-17p (pmol) 0 0 100 200 300 0 100 200 300 (min) (min)

Fig. 2. Time course of [3H]GDP and WT-Arf1p 0.6 arf1-17p [35S]GTP␥S binding to myr-Arf1p 0.3 γ GTPγS23˚C GTP S23˚C and myr-arf1-17p. Purified Arf1p or 0.5 arf1-17p (1.25 ␮M each) was mixed ␮ 3 0.4 with 10 M [ H]GDP (circle) or 0.2 GTPγS30˚C [35S]GTP␥S (triangle) at 23°C (closed 0.3 circles and triangles) and 30°C (open GTPγS30˚C circles and triangles). At appropriate

0.1 0.2 time intervals, aliquots of the reaction mixture were filtrated through a 0.1 nitrocellulose membrane filter and then the radioactivity of the bound Bound nucleotide (pmol) / arf1-17p (pmol) Bound nucleotide Bound (pmol) (pmol) / Arf1p 0 0 0 100 200 300 0 100 200 300 nucleotide was measured by (min) (min) scintillation counting. Roles of the noncatalytic region of ArfGAPs 2607

Fig. 3. Interactions of mutant arf1p with Arf GAPs in the DupLEX-ATM two-hybrid system. The interactions between N-terminally truncated (N17) Arf1p in the bait vector (pGlida) and Glo3p or Gcs1p in the prey vector (pJG4-5) were tested in the host strain EGY48. The Q71L mutation had been introduced in wild-type (ARF1), arf1-16 mutant (arf1-16) and arf1-17 mutant (arf1-17) genes. Transformants were plated on MVD (–Trp, –His), grown for 2 days, replicated on MVGal (minimal galactose medium) (–Trp, –His, –Leu), MVD (–Trp, –His), and MVD (–Trp, –His, –Leu) plates, and then further incubated at 30°C for 6 days. Empty prey vector (pJG4-5) was used as a negative control.

Gcs1p on arf1-16p/17p in vivo? Analysis of the amino acid 80 sequences of Glo3p and Gcs1p reveals 48% similarity and 28% 70 WT-Arf1p identity (Poon et al., 1999). The homology between Glo3p and Gcs1p is limited to their N-terminal region containing the 60 ArfGAP domain, which is known as the minimal domain to 50 execute the ArfGAP activity of rat Arfgap1 in vitro (Cukierman et al., 1995). By contrast, the similarity between 40 Glo3p and Gcs1p is very low in other regions of the molecules. 30 It has also been reported that the C-terminal non-catalytic

20 domain of rat Arfgap1 is required for its recruitment from GTP hydrolysis (%) GTP hydrolysis cytoplasm to membranes and for its interaction with the KDEL 10 receptor (Aoe et al., 1999; Huber et al., 1998). From these

0 aspects, we suspected that the clue to understand the difference 0204060 between Glo3p and Gcs1p in vivo lies in the C-terminal Time (min) domains of the proteins.

90 To examine this possibility, we performed domain swapping

Journal of Cell Science of Glo3p and Gcs1p and examined the ability of the chimeric 80 arf1-17p molecules to suppress the growth defect of arf1-16 (Fig. 5). 70 The ArfGAP domain of yeast ArfGAPs contains a functionally 60 important C2C2H2 zinc-finger motif (Zhang et al., 1998), and

50 the similarity between Glo3p and Gcs1p is especially high around this motif. When the C-terminal large portion (residues 40 60-353) of Gcs1p was replaced by the C-terminal region 30 (residues 65-494) of Glo3p (named Gcs1-A-Glo3p: 1-60/65-

20 494), overexpression of the chimeric protein rescued the temperature sensitivity of arf1-16. However, the reverse GTP hydrolysis (%) GTP hydrolysis 10 chimeric construct, Glo3-A-Gcs1p (1-64/60-353), did not 0 show the suppression activity. Furthermore, the overexpression 0204060 of Glo3-A-Gcs1p compromised the ts growth of arf1-16, just Time (min) like Gcs1p itself. Similarly, chimeric proteins fused at a slightly further downstream position, Gcs1-N-Glo3p (1- Glo3p (23˚C) Gcs1p (23˚C) 111/122-494) and Glo3-N-Gcs1p (1-122/113-353), caused Glo3p (35˚C) Gcs1p (35˚C) suppression or aggravation of the ts growth, depending on which protein the C-terminus was derived from. These results no ArfGAP (23˚C) support the differentiated roles of the C-terminal region of no ArfGAP (35˚C) Glo3p and Gcs1p.

Fig. 4. Time course of Arf GAP activity of Glo3p and Gcs1p. 0.2 Tandem Glo3 motif at the C-terminus of Glo3p is ␮M recombinant Glo3p (circle) or Gcs1p (triangle) was incubated with 1 ␮M [α-32P]GTP-loaded WT-Arf1 (upper panel) or arf1-17 essential for the suppression of arf1-16 (lower panel) protein at 23°C (closed circle and triangle) and 35°C Because Glo3p and Gcs1p showed different effects in vivo (open circle and triangle). Aliquots were withdrawn at the indicated depending on their C-termini, we went on to examine whether times and subjected to the analysis of bound nucleotide by thin-layer ArfGAPs from other eukaryotic organisms show any particular chromatography. similarity to either of them. We searched genome databases of 2608 Journal of Cell Science 119 (12)

Homo sapiens, Mus musculus, Caenorhabditis elegans, we introduced mutations in this motif of Glo3p and tested its Drosophila melanogaster and Arabidopsis thaliana using the ability to suppress the growth defect of arf1-16 (Fig. 6B-D). whole length of amino-acid sequences of Glo3p and Gcs1p Residues in either the first or second repeat of ISSxxxFG were independently, and picked up ArfGAP(s) that showed high replaced by alanines and subjected to the suppression test. blast scores in each species. As shown in Table 1, completely Overexpression of the glo3-11 and glo3-12 proteins, in which separable subgroups could be defined, and we hitherto classify only one ISS sequence was mutated, partially suppressed the these ArfGAPs as Glo3-type and Gcs1-type, respectively. ts growth of arf1-16. When the both ISS sequences were Interestingly, multiple alignments of the Glo3-type ArfGAPs mutated (glo3-13 and 14), the suppression activity was revealed limited but very high sequence similarity at their C- completely lost. This result indicates that the Glo3 motif is termini (Fig. 6A). We call this conserved C-terminal region the critical for the in vivo function of Glo3p. Glo3 motif. The Glo3 motif generally contains two repeats of the ISSxxxFG sequence, with the exception of D. Discussion melanogaster and C. elegans proteins, in which one of the two We have been investigating how Arf1p plays multiple roles in repeats diverges. K386 and L411 of S. cerevisiae Glo3p in this living cells. arf1 ts mutants we isolated show a variety of motif are also well conserved among the Glo3-type ArfGAPs. transport defects and morphological alterations in an allele- In contrast, none of Gcs1-type ArfGAPs contain the Glo3 specific manner. To identify partner molecules that participate motif. Other types of ArfGAPs such as Age1p or Age2p of in individual functions of Arf1p, we have screened for yeast do not possess the Glo3 motif either. multicopy suppressors of the arf1 ts mutants. In the present To examine the physiological importance of the Glo3 motif, study, we have demonstrated that the overexpression of GLO3, one of the ArfGAPs, suppresses the ts growth of arf1-16 and arf1-17 mutants, which are classified into the same intragenic complementation group, whereas other ArfGAPs (GCS1, AGE1 and AGE2) do not. The overproduction of Gcs1p is inhibitory for the growth of arf1-16 and arf1-17 cells. This result raises the possibility that different ArfGAPs act differently on Arf1p to allow it to fulfill divergent functions. In the nucleotide-binding assay, the arf1-17 protein showed lower binding of GDP than GTP at 30°C. If this imbalance of GDP/GTP binding was a cause of the mutant phenotype of arf1-17 cells, it seemed reasonable that the overproduction of the Glo3p ArfGAP suppressed it. The difference in the

Journal of Cell Science suppression activity between Glo3p and Gcs1p might be explained by their different affinities to the mutant Arf1p. However, the GAP activities of Glo3p and Gcs1p on the arf1-17 protein turned out to be almost identical in vitro. The key to solve this

Fig. 5. (A) Sequence alignment of the N-terminal domains of Glo3p, Gcs1p and rat Arfgap1. The ClustalW algorithm was used to create the alignment. Asterisks indicates positions that have a single, fully conserved residue; colons and stops indicate that the stronger- and weaker- score groups is fully conserved, respectively. SAT motifs (C2C2H2) (Zhang et al., 1998) are underlined, and the ArfGAP domain of rat Arfgap1 (residues 1-136) (Cukierman et al., 1995; Goldberg, 1999) is shaded. (B) Construction of chimeric proteins of Glo3p and Gcs1p. (C) Suppression activities of Glo3p and Gcs1p chimeric proteins. arf1-16 cells were transformed with pNY24-GLO3 (Glo3p), pNY24-GCS1-A-GLO3 (Gcs1- A-Glo3p), pNY24-GCS1-N-GLO3 (Gcs1-N-Glo3p), pNY24-GLO3-N-GCS1 (Glo3-N-Gcs1p), pNY24- GLO3-A-GCS1 (Glo3-A-Gcs1p), and pNY24-GCS1 (Gcs1p), and the transformants were grown at 23°C. Cells were diluted, spotted on MCD (–Trp) plates, and incubated at the indicated temperatures for 2 days. Integrants of wild-type ARF1 (WT) and mutants transformed with pYO324 (vector) were used as controls. Roles of the noncatalytic region of ArfGAPs 2609

Table 1. Glo3-type and Gcs1-type ArfGAPs from various species ArfGAP Species Glo3 type Gcs1 type S. cerevisiae Glo3p Gcs1p H. sapiens ZNF289 Arfgap1 Arfgap3 M. musculus Zfp289 Arfgap1 Arfgap3 D. melanogaster CG6838 CG4237 C. elegans F07F6.4 K02B12.7 A. thaliana AT5G46750.1 AT3G53710.1 AT4G17890.1 AT2G37550.1 AT2G35210.1

apparent disparity was revealed to lie in the non-catalytic regions of ArfGAPs. All known ArfGAPs contain the ArfGAP domain, which executes the ArfGAP catalytic activity, but the amino-acid sequences in the rest of molecules are different from each other. When the non-catalytic C-terminal portions of Glo3p and Gcs1p were exchanged, their effects on arf1-16 were also switched, suggesting that this region is required for their individual functions in vivo. Interestingly, we found a novel motif at the C-terminus of Glo3p (residues 386-422 of Glo3p), which is conserved among Glo3-type ArfGAPs in many eukaryotes. We named this region the Glo3 motif. The characteristic feature of this motif is one or two repeats of the ISSxxxFG sequence. Point mutations of the conserved motif resulted in the loss of the suppression activity in arf1-16, indicating that the Glo3 motif is required for the proper activity of Glo3p in living cells. These Glo3-motif mutant proteins are

Journal of Cell Science supposed to retain the ArfGAP activity, because the N-terminal ArfGAP domain remains intact. Therefore, the activity of Glo3p is probably modulated by the third molecule(s) through the interaction with the Glo3 motif in vivo. arf1-16/17 mutant proteins presumably have a problem in interacting with such modulators. Fig. 6. (A) Alignment of C-terminal amino acid sequences from What could be the third molecule(s) then? Glo3p has been various Glo3-type Arf GAPs. The ClustalW algorithm was used to thought to play an important role in the Golgi-to-ER retrograde create the sequence alignment. Identical amino acid positions for transport in cooperation with the complex. Dogic et each Glo3-type ArfGAP are marked with asterisks (*). Sc, S. al. (Dogic et al., 1999) showed in an analysis of yeast cerevisiae; Hs, H. sapiens; Mm, M. musculus; Ce, C. elegans; Dm, disruptants of the ArfGAP genes that the deletion of GLO3 D. melanogaster; At, A. thaliana. Note that one or two repeats of alone resulted in a defect of the retrieval of dilysine-tagged ISSxxxFG sequences exist in every Glo3-type ArfGAP. K386 and L411 of Glo3p are also conserved (shaded box). (B) Mutational proteins from the Golgi. Glo3p was also reported to interact ␥ ␤Ј analysis of the Glo3 motif. Various amino acid mutations were with Sec21p ( -COP) and Sec27p ( -COP), whereas Gcs1p introduced into this motif of the chimeric protein 3HA-Glo3p. failed to bind to coatomer both in a two-hybrid assay and in (C) NYY16-1 cells (arf1-16) were transformed with pYO324 immunoprecipitation analysis (Eugster et al., 2000; Lewis et (vector), pNY14-3HA-GLO3 (CEN 3HA-GLO3), pNY24-3HA- al., 2004). The Glo3 motif at the C-terminus may be required GLO3 (2 ␮ 3HA-GLO3), pNY24-glo3-m2HA (2 ␮ 3HA-glo3-11), for the interaction with the coatomer complex. In an attempt pNY24-glo3-m1HA (2 ␮ 3HA-glo3-12), pNY24-glo3-m3HA (2 ␮ to support this possibility by a genetic analysis, however, none 3HA-glo3-13) and pNY24-glo3-m5HA (2 ␮ 3HA-glo3-14). Cells of the COPI genes could suppress the ts growth of arf1-16 by were grown, diluted and spotted on MCD (–Trp) plates, and their single overproduction (our unpublished results). incubated at the indicated temperatures for 2 days. Integrants of Another class of molecules we are interested in are Bet1p wild-type ARF1 (NYY0-1: WT) transformed with pYO324 were used as a control. (D) Immunoblotting of 3HA-Glo3p to examine and Bos1p. Recent studies have shown that the ArfGAP expression levels. The transformants that were used in C were grown activity is required not only to remove the protein coat but also to an early log phase at 23°C, and incubated at 35°C for 1 hour. Total for concentration of cargo into vesicles (Lanoix et al., 1999; cell lysates (60 ␮g) were prepared and analyzed by immunoblotting Lanoix et al., 2001; Nickel et al., 1998; Pepperkok et al., 2000). using the anti-HA monoclonal and anti-Sec61p antibodies. Lane In the case of a related GTPase, Sar1p, we have also shown numbers correspond to the transformant numbers used in C. 2610 Journal of Cell Science 119 (12)

that the regulation of GTP hydrolysis by Sec23p GAP is the present study. The overexpression of OLE1 may increase important for cargo selection during COPII vesicle budding the amount of conical lipids, resulting in the gain of more (Sato and Nakano, 2005). If Glo3p promotes the sorting of a binding sites for ArfGAPs. particular set of cargo molecules, such cargo could be isolated In summary, our results suggest that Glo3p and Gcs1p as multicopy suppressors of arf1-16. BET1 and BOS1, which possess different functions in vivo and that their non-catalytic have been isolated as multicopy suppressors of arf1-16/17 in C-terminal regions are required for their individual activities. the present study, encode SNARE molecules functioning Both Glo3-type and Gcs1-type ArfGAPs are found in human, during the vesicular recycling between the ER and the Golgi mouse, fly, worm and plants. The Glo3-type ArfGAPs may apparatus (Martinez-Menarguez et al., 1999; Rexach et al., well play distinct roles from Gcs1-type ArfGAPs, not only in 1994). BET1 and BOS1 were also isolated as multicopy yeast but also in other eukaryotic organisms. The human suppressors of the temperature sensitivity of ⌬glo3 mutant genome contains at least 16 ArfGAP proteins. They have been cells (Poon et al., 1999). Furthermore, Rein et al. (Rein et al., divided into several subgroups (Arfgap1 type, Gits type and 2002) reported that Glo3p enhances the binding of COPI and AZAP type) according to the overall domain structures arf1⌬N17p to the cytoplasmic tails of Bet1p, Bos1p and (Randazzo and Hirsch, 2004). Human Arfgap1 and Arfgap3 Sec22p. If one hypothesizes that arfGAP has a role to enrich are categorized as Arfgap1-subgroup proteins. In this study, we these SNAREs as cargo into COPI vesicles, then the defects of present the evidence that the Arfgap1-subgroup proteins are arf1-16/17 mutant proteins could be explained by the defective further classified into two groups, Gcs1 type and Glo3 type. ability of responsible ArfGAP to select cargo. In our The Glo3-type ArfGAPs have the Glo3 motif in their non- preliminary experiment, arf1-16p/17p showed rather elevated catalytic domains. binding to Bet1p than the wild-type Arf1p, but this binding was During the preparation of this manuscript, Lee et al. (Lee et not significantly affected by the addition of either Glo3p or al., 2005) published a paper reporting that rat Arfgap1 contains Gcs1p (our unpublished result). at least two distinct binding sites for coatomer, one in its Why does Gcs1p exaggerate the ts defect of arf1-16 and catalytic domain and the other in the non-catalytic domain. arf1-17 cells? Glo3p and Gcs1p are known to play essential They also revealed that a truncated Arfgap1 containing the first overlapping functions in retrograde vesicular transport (Poon 136 residues (out of non-catalytic C-terminal domain) failed to et al., 1999). Indeed, glo3⌬ gcs1⌬ double deletion mutant cells bind cargo proteins. The ALPS motif was also found in the are inviable (Poon et al., 1999; Zhang et al., 1998). However, non-catalytic regions of rat Arfgap1 and Gcs1p (Bigay et al., there is also evidence suggesting that Glo3p and Gcs1p have 2005). Further experiments should address the physiological additional and distinct roles. For example, the function of roles of non-catalytic domains of ArfGAPs, including the Glo3 Gcs1p overlaps with Age2p for transport from the yeast trans- motif. Arf1p probably uses these different kinds of ArfGAPs Golgi network (Poon et al., 2001b). The simultaneous and thus plays multiple roles in living cells. overexpression of GLO3 and GCS1 resulted in weaker suppression of arf1-16/17 than that of GLO3 alone (data not Materials and Methods shown), suggesting that these ArfGAPs are competing for Strains and plasmids Saccharomyces cerevisiae strains, NYY0-1 (WT), NYY16-1 (arf1-16) and NYY17- Journal of Cell Science something. Arf1p itself is not a likely candidate because the 1 (arf1-17), were described previously (Yahara et al., 2001). The yeast single-copy overexpression of other ArfGAPs, AGE1 and AGE2, have no plasmid, pRS314, and multicopy plasmids, pYO324 and pYO325, have been effect on the growth of the mutants. One attractive model may described elsewhere (Ohya et al., 1991; Sikorski and Hieter, 1989). GCS1, AGE1 be that Glo3p and Gcs1p function on the same membrane and and AGE2 genes were amplified from genomic DNA by PCR using gene-specific primers. After the PCR fragments were cloned into plasmids, the regions of ORF compete with each other for the binding site on the membrane. and ~500 bp each of 5Ј upstream and 3Ј downstream were re-obtained by Antonny’s group showed that rat Arfgap1 and Gcs1p bind homologous recombination. For GLO3, we subcloned the gene from one of the preferentially to liposomes containing conical lipids but not multicopy suppressor clones of arf1-16. The DNA fragments we cloned into the those of cylindrical lipids, and the preference for conical lipids pYO324 plasmid were: GLO3, upstream 799 bp/ORF 1482 bp/downstream 622 bp; GCS1, upstream 871 bp/ORF 1059 bp/downstream 1593 bp; AGE1, upstream 941 correlated with its GAP activity on liposome-bound Arf1p- bp/ORF 1449 bp/downstream 388 bp; and AGE2, upstream 676 bp/ORF 897 GTP (Antonny et al., 1997; Bigay et al., 2003). More recently, bp/downstream 241 bp. They were named pNY24-GLO3, pNY24-GCS1, pNY24- they proposed that the central region of Arfgap1 contains a AGE1 and pNY24-AGE2, respectively. The epitope-tagged 3HA-GLO3 and GCS1- 3HA were constructed as follows. Site-directed mutagenesis was used to create one sequence that acts as a lipid packing sensor and named it the SpeI site adjacent to the start codon of GLO3 in pNY24-GLO3 and another SpeI ALPS motif (Bigay et al., 2005). Because this ALPS motif is site just before the stop codon of GCS1 in pNY24-GCS1. The NheI-NheI fragment well conserved in the Gcs1-type ArfGAPs but not in others, it from pYT11 (Takita et al., 1995) containing three tandem copies of the hemagglutinin epitope (HA) was inserted at these SpeI sites. The resulting plasmids, remains an open question whether or not the Glo3-type pNY24-3HA-GLO3 and pNY24-GCS1-3HA, were confirmed for functionality by ArfGAPs also sense lipid structures. Since Glo3p possesses their effects on arf1-16 (supplementary material Fig. S1). To make chimeric overlapping functions with Gcs1p in retrograde transport from constructs of Glo3p and Gcs1p, ApaLI or NruI sites were introduced in appropriate the Golgi to the ER, but not with Age1 and Age2p (Poon et regions of GLO3 and GCS1. BglII-ApaLI and BglII-NruI regions of GLO3 plasmid were replaced by SacI-ApaLI and SacI-NruI fragments of GCS1, and the resulting al., 1999; Poon et al., 2001b), it may be reasonable to assume constructs expressed residues 1-59 of Gcs1p fused to 65-494 of Glo3p (pNY24- that Glo3p and Gcs1p function on the same membrane, e.g. GCS1-A-GLO3) and residues 1-111 of Gcs1p fused to 122-494 of Glo3p (pNY24- Golgi. An excess amount of Gcs1p might fill up the binding GCS1-N-GLO3) under the GCS1 promoter, respectively. EcoRI-NruI and EcoRI- ApaLI regions of GCS1 plasmid were also replaced by EcoRI-NruI and EcoRI- sites of ArfGAPs on the membrane where Glo3p also binds. ApaLI fragments of GLO3, and the resulting constructs expressed residues 1-122 The critical defect of the arf1-17 mutant protein could be an of Glo3p fused to 113-353 of Gcs1p (pNY24-GLO3-N-GCS1), and residues 1-64 inability to bind specific cargo, e.g. Bet1p, which needs access of Glo3p fused to 60-353 of Gcs1p (pNY24-GLO3-A-GCS1) under the GLO3 to Glo3p on the membrane. OLE1, a gene encoding a fatty acid promoter, respectively. In all constructions, DNA sequences were confirmed. desaturase required for monounsaturated fatty acid synthesis, Isolation of multicopy suppressors of arf1-16 has also been isolated as a multicopy suppressor of arf1-16 in The S. cerevisiae genomic DNA library based on YEp13, a multicopy vector with Roles of the noncatalytic region of ArfGAPs 2611

the LEU2 marker (Yoshihisa and Anraku, 1989), was introduced into the arf1-16 The [32P]GTP-loaded Arf1 proteins were diluted into the assay system containing mutant (NYY16-1). Transformants were grown on MVD (minimal glucose 4.6 mM MgCl2, 50 mM HEPES-KOH (pH 7.5), and 10 mM ATP. Following a brief medium) lacking leucine at 23°C for 1 day and then incubated at 35°C for 3 days. warming, the reaction was initiated by the addition 0.2 ␮M ArfGAP proteins. At Colonies that grew at 35°C were re-streaked on MVD plates and confirmed for appropriate times, aliquots were transferred into microfuge tubes containing the growth at 35°C. Plasmids were recovered from the candidate clones and re- final concentration of 250 mM EDTA. The samples were spotted onto a introduced into arf1-16 to verify the suppression activity. Out of approximately polyethyleneimine cellulose plate (MERCK) and developed with 1 M LiCl/1 M 50,000 transformants screened, 78 positive clones were obtained. Among strong HCOOH (1:1, v/v). The plate was dried, fluorographed, and quantitatively analyzed suppressors, four clones contained either ARF1 or ARF2. In addition, we found that with an image analyzer BAS2500 (Fuji Film, Tokyo, Japan). 12, 6, 4, 14 and 2 clones repeatedly contained genomic fragments harboring GLO3, BET1, BOS1, OLE1 and GYP6, respectively. They were subcloned into pYO325, a Databases multicopy vector with the LEU2 gene, to confirm that the suppression activities To search homologues of Glo3p and Gcs1p, we used the Mouse Genome Database, depended on these five genes. Mouse Genome Informatics Web Site, The Jackson Laboratory, Bar Harbor, Maine (http://www.informatics.jax.org) for Mus musculus (Blake et al., 2003; Eppig et al., Two-hybrid assay 2002), the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp) for Homo sapiens The bait plasmid pGlida, the prey plasmid pJG4-5, and the yeast reporter strain (Miyazaki et al., 2003), FlyBase (http://flybase.bio.indiana.edu) for Drosophila EGY48 were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). melanogaster (FlyBaseConsortium, 2003), WormBase (http://www.wormbase.org) Plasmid constructions and the examination of interaction between for Caenorhabditis elegans and the Arabidopsis Information Resource (⌬N17)Arf1(Q71L)p and ArfGAPs were performed as previously described except (http://www.arabidopsis.org) for Arabidopsis thaliana on Nov. 20, 2004. Multiple that the bait pEG202 was replaced with pGlida (Eugster et al., 2000). alignment of sequences was done using the ClustalW system of ClustalW 1.8.3 program (http://www.ddbj.nig.ac.jp/search/ex_clustalw-e.html) (Thompson et al., Purification of myristoylated-Arf1 proteins 1994). Yeast N-myristoyltransferase (NMT1) expression vector (pACYC177-ET3d-NMT1) was constructed as previously described (Haun et al., 1993) and its activity was Immunoblotting and antibodies confirmed using [1-14C]myristic acid (data not shown). The full-length ARF1 and Cells were cultured to a middle logarithmic phase. Total cell lysates were prepared arf1-17 genes were cloned into NdeI-XhoI sites of pET21a, and E. coli BL21 (DE3) by agitation with glass beads in 62.5 mM Tris-HCl (pH 6.8), 2.35% SDS, 10% (w/v) was co-transformed with the resulting plasmids (pET-ARF1 and pET-arf1-17, glycerol, and 1 mM PMSF, and treated at 65°C for 10 minutes. They were subjected respectively) and pACYC177-ET3d-NMT1. Transformed cells were selected for to immunoblotting by standard procedures and visualized by the ECL-plus both ampicillin and kanamycin resistance and grown at 37°C. After myristate (50 chemiluminescent detection system (Amersham). The anti-Arf1p antibody was ␮M) bound to BSA (6 ␮M) was added to the culture (Franco et al., 1995), the described previously (Yahara et al., 2001). The anti-Sec61p antibody was a gift from coexpression of ARF1 and NMT1 was induced with 1 mM isopropyl ␤–D- R. Schekman. Monoclonal anti-HA antibodies, HA7 and 12CA5, were obtained thiogalactoside (IPTG), and the temperature was reduced to 30°C to increase the from Sigma (MO, USA) and Roche (Basel, Switzerland), respectively. yield of myristoylation. Purification of myristoylated-Arf1 and myristoylated-Arf1- 17 proteins was performed by the method previously described with some This work was supported by Grants-in-Aid from the Ministry of modifications (Peyroche and Jackson, 2001; Randazzo et al., 1995; Weiss et al., Education, Science, Sports and Culture of Japan and by a fund from 1989). Briefly, the cells were harvested, resuspended in 50 ml lysis buffer [20 mM the Bioarchitect Research Project of RIKEN. Tris-HCl (pH 8), 1 mM EDTA, 200 ␮M GDP, 1 mM DTT and 0.4 mM PMSF], and lysed. After removal of debris, the lysate was centrifuged at 100,000 g for 1 hour at 4°C. The supernatant was recovered, and ammonium sulfate was added to 33% References saturation. The mixture was incubated for >1 hour at 4°C with agitation and Antonny, B., Huber, I., Paris, S., Chabre, M. and Cassel, D. (1997). Activation of ADP- centrifuged at 20,000 g for 30 minutes at 4°C. The pellets were resuspended in ribosylation factor 1 GTPase-activating protein by phosphatidylcholine-derived dialysis buffer [20 mM Tris-HCl (pH 8), 1 mM MgCl2, 5 ␮M GDP, 1 mM DTT diacylglycerols. J. Biol. Chem. 272, 30848-30851. and 0.4 mM PMSF], and dialyzed against the same buffer three times (2 hours, Aoe, T., Huber, I., Vasudevan, C., Watkins, S. C., Romero, G., Cassel, D. and Hsu, overnight, then 3 hours). The dialyzed supernatant was mixed with DEAE- V. W. (1999). The KDEL receptor regulates a GTPase-activating protein for ADP- ribosylation factor 1 by interacting with its non-catalytic domain. J. Biol. Chem. 274, Sepharose FF (Amersham), incubated for 10 minutes at 4°C, and centrifuged at

Journal of Cell Science 20545-20549. 30,000 g for 10 minutes. The material that failed to bind to DEAE-Sepharose was Bigay, J., Gounon, P., Robineau, S. and Antonny, B. (2003). Lipid packing sensed by concentrated to about 4 ml by ultrafiltration using a Centriprep 10 (Amicon). The ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 426, ␮ supernatant was filtrated through a 0.45 m filter, and then loaded onto a 120-ml 563-566. HiLoad 16/60 Superdex 200 pg column (Amersham) equilibrated in buffer A (20 Bigay, J., Casella, J.-F., Drin, G., Mesmin, B. and Antonny, B. (2005). ArfGAP1 mM Tris-HCl, pH 8, 1 mM MgCl2 and 1 mM DTT). responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24, 2244-2253. Guanine-nucleotide binding assay Blake, J. A., Richardson, J. E., Bult, C. J., Kadin, J. A., Eppig, J. T. and The Mouse GTP␥S- and GDP-binding activities were measured by the procedure described Genome Database Group (2003). MGD: the mouse genome database. Nucleic Acids previously with some modifications (Peyroche and Jackson, 2001; Randazzo et al., Res. 31, 193-195. 1995). Purified Arf1p or arf1-17p (1.25 ␮M each) was incubated in 25 mM HEPES- Cukierman, E., Huber, I., Rotman, M. and Cassel, D. (1995). The ARF1 GTPase- activating protein: zinc finger motif and Golgi complex localization. Science 270, 1999- KOH (pH 7.5), 1 mM DTT, 1 mM MgCl2, 3 mM DMPC, 0.1% (w/v) sodium cholate and 10 ␮M [3H]GDP or [35S]GTP␥S (10,000 cpm/pmol) at 23 or 30°C. At 2002. Dogic, D., de Chassey, B., Pick, E., Cassel, D., Lefkir, Y., Hennecke, S., Cosson, P. appropriate time intervals, aliquots of the reaction mixture were taken and the ␮ and Letourneur, F. (1999). The ADP-ribosylation factor GTPase-activating protein reaction was stopped by diluting samples (20 l) into 1 ml of stop buffer [20 mM Glo3p is involved in ER retrieval. Eur. J. Cell Biol. 78, 305-310. HEPES-KOH (pH 7.5), 100 mM NaCl and 10 mM MgCl2] at 4°C. Proteins were Donaldson, J. G. and Jackson, C. L. (2000). Regulators and effectors of the ARF filtrated through a nitrocellulose membrane filter, washed four times with 2 ml of GTPases. Curr. Opin. Cell Biol. 12, 475-482. the stop buffer and then the radioactivity of the bound nucleotide was measured by Donaldson, J. G., Honda, A. and Weigert, R. (2005). Multiple activities for Arf1 at the scintillation counting. Golgi complex. Biochim. Biophys. Acta Mol. Cell Res. 1744, 364-373. Eppig, J. T., Blake, J. A., Burkhart, D. L., Goldsmith, C. W., Lutz, C. M. and Smith, GAP assays C. L. (2002). Corralling conditional mutations: a unified resource for mouse Construction of expression vectors encoding hexahistidine (His6)-tagged GCS1 and phenotypes. Genesis 32, 63-65. GLO3 was performed as described previously (Poon et al., 2001a). The plasmid Eugster, A., Frigerio, G., Dale, M. and Duden, R. (2000). COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP. EMBO J. 19, 3905- pGLO3-HIS encoding His6-tagged GLO3 at its C-terminus was constructed by cloning the gene into pET21a (Novagen). For GCS1, the first six codons of the GCS1 3917. ORF were replaced by His sequences, and cloned into pET21a. The resulting FlyBaseConsortium (2003). The FlyBase database of the Drosophila genome projects 6 and community literature. Nucleic Acids Res. 31, 172-175. plasmid, pHIS-GCS1, expresses N-terminally His -tagged GCS1. Both Gcs1p and 6 Franco, M., Chardin, P., Chabre, M. and Paris, S. (1995). Myristoylation of ADP- Glo3p were largely insoluble and were thus prepared under denaturing conditions ribosylation factor 1 facilitates nucleotide exchange at physiological Mg[IMAGE] by guanidine extraction followed by Ni-NTA purification, as described before, levels. J. Biol. Chem. 270, 1337-1341. except that a HiTrap Q (Amersham) column was used instead of a Resource Q Goldberg, J. (1999). Structural and functional analysis of the ARF1-ARFGAP complex column. GAP activity was assayed as described before with some modifications reveals a role for coatomer in GTP hydrolysis. Cell 96, 893-902. (Cukierman et al., 1995; Huber et al., 2001). Arf1 protein (5 ␮M) was first incubated Haun, R. S., Tsai, S. C., Adamik, R., Moss, J. and Vaughan, M. (1993). Effect of for 15 minutes at 23°C with 2 ␮M [32P]GTP in the presence of 50 mM HEPES- myristoylation on GTP-dependent binding of ADP-ribosylation factor to Golgi. J. Biol. KOH (pH 7.5), 1 mM DTT, 2 mM EDTA, 1 mM MgCl2, 10 mM ATP, 3 mM DMPC Chem. 268, 7064-7068. and 0.1% cholate. The reaction was terminated by the addition of MgCl2 to 2 mM. Huber, I., Cukierman, E., Rotman, M., Aoe, T., Hsu, V. W. and Cassel, D. (1998). 2612 Journal of Cell Science 119 (12)

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