It has been demonstrated that Saccharomyces cerevisiae Vam6p/Vps39p plays a critical role in the tethering steps of vacuolar membrane fusion by facilitating guanine nucleotide exchange on small guanosine triphosphatase (GTPase) Vam4p/Ypt7p. We report here the identification and characterization of a novel protein in Aspergillus nidulans, AvaB, that exhibits similarity to Vam6p/Vps39p and plays a critical role in vacuolar morphogenesis in A. nidulans. AvaB is comprised of 1058 amino acids with amino-terminal citron homology (CNH) and central clathrin homology (CLH) domains, as observed for other Vam6p/Vps39p family proteins. Disruption of avaB in A. nidulans resulted in the fragmentation of vacuoles and reduced growth rate under various growth conditions, implying its importance in maintaining vacuolar morphology and function. Yeast two-hybrid analysis demonstrated the interaction of AvaB with AvaA, a Vam4p/Ypt7p homolog in A. nidulans, as well as the homooligomer formation of AvaB, suggesting that AvaB performs its function through hetero- or homophilic protein–protein interactions.
Most proteins destined to the vacuoles, the organelle functionally equivalent to mammalian lysosomes, are first targeted to the endoplasmic reticulum, after which they are sorted in the late compartment of the Golgi apparatus from other proteins of the secretory pathway, and are delivered to the vacuole in membrane vesicles that eventually fuse with the vacuoles [1,2]. The sorting processes are accomplished through coordinated actions of more than 40 gene products which have been identified by genetic screens in Saccharomyces cerevisiae for mutants defective in vacuolar protein sorting (vps) [3–6], vacuolar proteinase activity (pep) , vacuole segregation (vac)  and in vacuolar morphogenesis (vam) . These mutants are classified into six subgroups (classes A–F) based on the defects they display [10,11], and the function of responsible gene products has been elucidated through biochemical as well as molecular biological analyses. In particular, in vitro reconstitution of vacuolar membrane fusion has been utilized to dissect each single step of vesicle targeting and fusion, demonstrating that the ordered events of priming, tethering, docking, and fusion mediated by VAM/VPS gene products ensure the correct and efficient sorting of vacuolar proteins and vacuolar inheritance .
Class B vps mutants are characterized by the lack of large central vacuoles and accumulation of fragmented endosomal compartments, as well as missorting of vacuolar hydrolases such as carboxypeptidase Y and alkaline phosphatase. Recent advances in the study of vacuolar membrane fusion have elucidated that the products of class B VPS genes, VAM2/VPS41, VAM6/VPS39 and YPT7, are involved in the tethering stage of vacuolar homotypic membrane fusion [13–17]. In the process of vacuole homotypic fusion, following the priming stage that is driven by Sec18p ATPase to disassemble cis-SNARE complex, vacuoles to be fused need to be tethered in close proximity with each other so that the trans-SNARE complex formation is allowed. The tethering complex also includes class C Vps proteins (Vps11p, Vps18p, Vps16p, and Vps33p) which are thought to be symmetrically localized on both of the apposing membranes. In this complex, Vam6p/Vps39p stimulates guanine nucleotide exchange on small guanosine triphosphatase (GTPase) Vam4p/Ypt7p and activates it, which in turn plays a pivotal role in tethering through the association with class C Vps complex [18,19].
Compared to S. cerevisiae, much less is known as for the physiological function of vacuoles in filamentous fungi, albeit its potential importance in maintaining cellular homeostasis during growth and differentiation. In order to understand the vacuolar function in filamentous fungi, we previously isolated and characterized genes, vpsA and avaA, that are involved in vacuolar protein sorting, maintenance of vacuolar morphology and function in Aspergillus nidulans, as homologs of VPS1 and VAM4/YPT7 of S. cerevisiae, respectively [20,21]. In this study, we report the molecular cloning and characterization of avaB, a VAM6/VPS39 homolog in A. nidulans. AvaB protein shares moderate similarity with Vam6p/Vps39p homologs from various species, and contains citron homology (CNH) and clathrin homology (CLH) domains in the N- and C-terminal regions, respectively. Disruption of avaB in A. nidulans resulted in fragmentation of vacuoles and reduced growth in various media. AvaB is involved in the heterologous (with AvaA) and homologous (with AvaB itself) protein–protein interactions, which is the first experimental demonstration that a Vam6p/Vps39p family protein interacts in both ways. These results suggest a role for AvaB in the homotypic fusion of vacuolar membranes through interaction with class B VPS gene products in A. nidulans.
Materials and methods
A. nidulans strains, growth conditions and transformation
A. nidulans FGSC A26 (biA1 veA1) was used as a control strain. For the transformation, A. nidulans strains FGSC A89 (biA1 argB2) and ABPU1 (pyrG89 biA1 wA3 argB2 pyroA4) were used. For the observation of vacuole structures by expressing CpyA-EGFP (enhanced green fluorescent protein) (in which S. cerevisiae carboxypeptidase Y-like protein from A. nidulans, CpyA , is fused to EGFP), A. nidulans strain CGEBG8 was generated by transforming ABPU1 with pCGEBG, harboring cpyA-egfp fusion gene that is driven by Aspergillus oryzae pgkA promoter, and A. nidulans pygG gene [22,23]. Complete medium (YG) and supplemented minimal medium (MM) were used for the growth of A. nidulans. Standard procedures were employed for the transformation of A. nidulans.
Cloning and sequencing of A. nidulans avaB gene
The search for S. cerevisiae VAM6/VPS39 homolog gene in A. nidulans was performed by comparing the Vam6p/Vps39p sequence against the A. nidulans Expressed Sequence Tag Database (Advanced Center for Genome Technology, University of Oklahoma; http://www.genome.ou.edu/fungal.html). This search indicated the presence of a Vam6p/Vps39p-like sequence (m6b12a1.r1) which was used to design primers M6-1 (5′-ggaattctgatagtcaagctcaggctg-3′) and M6-2 (5′-ggaattccttctttccgtacagaaagtc-3′). Polymerase chain reaction (PCR) amplified a 0.6-kb DNA fragment, the nucleotide sequence analysis of which confirmed that the fragment encoded VAM6/VPS39 homolog. The same set of primers was used to screen A. nidulans genomic cosmid library (purchased from Fungal Genetics Stock Center, USA) by colony PCR. One of the clones, W09A04, was later found to contain full-length VAM6/VPS39 homolog sequence and thus further analyzed. Southern blot analysis using the 0.6-kb DNA fragment as a probe demonstrated that the gene was carried on 4-kb BamHI and 3-kb EcoRI fragments (Fig. 2). These DNA fragments were isolated and subcloned into the corresponding sites of pBluescript II SK(+), generating pBB and pEE, respectively. The nucleotide sequence of inserts was determined. The A. nidulans VAM6/VPS39 homolog was referred to as avaB (A. nidulans vacuolar morphology).
The avaB cDNA was isolated by PCR using the A. nidulans cDNA library as a template and the primer M6-N which contains putative start codon and the downstream primer M6-C1. Their sequences are: M6-N, 5′-agccATGctctccgcattca-3′ (uppercase letters indicate start codon); M6-C1, 5′-ggtctagactatcacaccaagtc-3′. By comparing the genomic and complementary DNA sequences, the positions of three introns were determined as indicated in Fig. 1.
avaB gene disruption in A. nidulans
For the disruption of avaB gene in A. nidulans, the following procedure was employed. pBB which contains the 4-kb BamHI fragment carrying most of the coding region of avaB in pBluescript II SK(+) was partially digested with BglII at one of the two BglII sites present in avaB gene, and the resultant 7-kb DNA fragment was isolated. This fragment was ligated with the 2.2-kb BamHI fragment carrying argB gene from pYARG , generating argB-disrupted avaB gene in which argB was inserted into the central BglII site of avaB. The resultant plasmid was digested with BamHI and the 6.2-kb DNA fragment containing argB-disrupted avaB gene was used to transform A. nidulans A89 strain. Transformants were selected on MM plates and out of 26 transformants obtained, a transformant named D12 was selected as the authentic avaB-disrupted strain. Disruption of avaB gene in D12 was confirmed by PCR using primer sets M6-1 and M6-2, which should generate 0.6-kb (control) and 2.8-kb (ΔavaB) DNA fragments, or M6-N (5′-agccatgctctccgcattca-3′) and M6-2, which should generate 2.0-kb (control) and 4.2-kb (ΔavaB) DNA fragments. Southern blot analysis was performed by digesting chromosomal DNA from A26 and D12 strains with PstI or BglII and probing with 0.4-kb BamHI/XhoI fragment of avaB gene, which should generate signals of 2.3-kb (PstI) or 1.3- and 7-kb (BglII) bands for control, and 1.8-kb (PstI) or 2.5- and 7-kb (BglII) bands for ΔavaB, respectively. For the disruption of avaB in CGEBG8 strain expressing CpyA-EGFP, a similar experimental strategy was employed, and a transformant named CGABD5 was selected.
Staining and microscopic observation of vacuoles
Vacuolar staining by CFDA (5-(and 6-)carboxy-2′,7′-dichlorofluorescein diacetate) was done as follows. After growing cells in liquid MM media for 12 h at 37°C, 1/10 and 1/1000 volumes of 500 mM citrate-NaOH (pH 3.0) and 10 mM CFDA in dimethylformamide, respectively, were added. Cells were incubated for 30 min at 37°C, after which cells were washed and observed. Morphology and fluorescence of cells were observed and photographed using a BX52 fluorescence microscope (Olympus, Japan) equipped with a SenSys-1401E cooled CCD camera (Roper Scientific, USA).
Plasmids pGAD-C1, pGBD-C1, and the host strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δgal80ΔLYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) for yeast two-hybrid analysis were generous gifts from Dr. Elizabeth A. Craig . The full-length avaB cDNA for the two-hybrid analysis was obtained by PCR using the primers AVBS-F and AVBS-R (AVBS-F: 5′-ggcccgggATGctctccgcattca-3′; AVBS-R, 5′-ggcccgggcgatactaTCAcgaag-3′; uppercase letters and underlines indicate start/stop codons and SmaI sites, respectively). The resultant DNA fragment was digested with SmaI, and ligated in-frame to SmaI-digested pGAD-C1 and pGBD-C1, generating pGAD-AB and pGBD-AB, respectively. The 0.6-kb EcoRI fragment carrying avaA cDNA was excised from pCAV , blunt-ended and ligated to SmaI-digested pGAD-C1, generating pGAD-AA. Cells transformed with either one of these plasmids or cotransformed with the combination of plasmids were selected on SD containing 20 μg ml−1 each of histidine, adenine and uracil and 10 μg ml−1 methionine. For testing AvaB homooligomeric or AvaA–AvaB heterooligomeric interactions, growth of transformants in the media deficient for either histidine (in the presence of 1 mM 3-aminotriazole) or adenine was examined.
Results and discussion
Isolation of genomic and complementary DNAs encoding VAM6/VPS39 homolog from A. nidulans
By searching the A. nidulans Expressed Sequence Tag Database for sequences that show similarity to S. cerevisiae VAM6/VPS39 using the BLAST algorithm, we identified a cDNA sequence of an approximate length of 610 bp. Based on this sequence, we designed a set of primers, M6-1 and M6-2, which was used for the PCR amplification of the corresponding fragment using A. nidulans genomic DNA as a template. A 0.6-kb DNA fragment was amplified, and the nucleotide sequence analysis demonstrated that the sequence was identical to that found in the database. The same set of primers was used to screen A. nidulans genomic cosmid DNA library by PCR, which identified a single clone (W09A04) carrying the entire coding region of VAM6/VPS39 homolog. The VAM6/VPS39 homolog gene was designated as avaB and further analyzed. The corresponding cDNA fragment was isolated by PCR as described in Section 2.2, and the nucleotide sequences of genomic and complementary DNAs were compared to locate the position of introns.
The avaB gene is comprised of 3323 bp including three introns, encoding a protein of 1058 amino acids with a calculated molecular mass of 117.8 kDa (Fig. 1A). No typical TATA nor polyadenylation signal sequences were found in the 5′-upstream and 3′-downstream regions, respectively. The amino acid sequence of AvaB shares approximately 30% identity with Vam6p homologs from various species throughout evolution, among which the homology with putative Vam6p from Schizosaccharomyces pombe is the highest. Similarity with S. cerevisiae Vam6p/Vps39p was observed only in the C-terminal half of AvaB (25%).
Searches for domain structures of AvaB were done utilizing the Pfam HMM database (http://pfam.wustl.edu/hmmsearch.shtml) and identified the N-terminal CNH domain (residues 20–365; Fig. 1B) and the central CLH domains (residues 644–673 and 743–929). While the CNH domain has been proposed to be involved in interaction with small GTPases [18,26,27], the CLH domain has been shown to participate in the homo- or heterophilic protein–protein interactions [27–29]. These observations suggest that the AvaB protein interacts with the A. nidulans homolog of Vam4p/Ypt7p small GTP binding protein (AvaA) and activates it, as well as with the AvaB protein itself, forming hetero- and homooligomers. These possibilities were examined by the yeast two-hybrid analysis (see below).
To test if avaB is able to complement the absence of VAM6/VPS39 gene in S. cerevisiae, avaB cDNA was expressed in Δvam6/vps39 strain under the control of inducible GAL1 promoter. However, the fragmentation of vacuoles, a phenotype of Δvam6/vps39 strain , was not complemented in neither of glucose nor galactose media, indicating that avaB failed to replace the function of VAM6/VPS39 in S. cerevisiae (data not shown).
Disruption of the avaB gene
To examine the involvement of AvaB in vacuolar biogenesis, we generated a strain disrupted for the avaB gene in A. nidulans. For this purpose, a 6.2-kb BamHI fragment carrying avaB gene interrupted by argB selectable marker was generated (Fig. 2A) and transformed to A. nidulans A89 strain as described in Section 2.3. PCR amplification of the genomic DNA from the control (A26) and putative argB-disrupted strain, D12, displayed that the insertion of a 2.2-kb argB marker in the avaB allele had occurred in D12 (Fig. 2B). This was further confirmed by Southern blot analysis (Fig. 2C). In the following experiments, phenotypes of ΔavaB strain D12 were compared with that of the control strain A26.
The growth of control and D12 strains incubated for 3 days on agar plates was compared under various media conditions (Fig. 3). Compared with the control strain A. nidulans A26, D12 grew slowly irrespective of the nutrient conditions, i.e. when grown on YG, 1/2YG and MM plates. The growth of D12 on MM adjusted at pH 4, 7, or 11, or MM containing 1.2 M NaCl, 1.2 M sorbitol, G418 (200 μg ml−1), or cycloheximide (200 μg ml−1) was similarly retarded. Thus, the disruption of avaB in A. nidulans caused retarded growth irrespective of the media constituents.
Vacuolar morphology of control and D12 strains of A. nidulans was observed by Nomarski optics and fluorescence microscopy after staining the cells with CFDA (Fig. 4A). In the control strain, vacuoles stained with CFDA were observed as oval structures aligned in the hyphae with regular intervals (Fig. 4A, b). These structures colocalized with ‘dents’ visualized by Nomarski optics (compare Fig. 4A, a and b). In the ΔavaB strain, however, no prominent vacuole-like structures were observed either by CFDA staining or by Nomarski optics, and only small dots with weak fluorescence were visualized, indicating that the vacuoles dispersed as fragments in the cytoplasm (Fig. 4A, c and d). When the vacuolar protein of A. nidulans, CpyA , which is a counterpart of S. cerevisiae carboxypeptidase Y, was fused to the amino-terminus of EGFP and expressed in ΔavaB, the fluorescence was again observed as small dots that were distributed throughout the hyphae (Fig. 4B, d), in contrast to the fluorescence in typical vacuole-like structures in the control strain (Fig. 4B, b). In addition, the overall fluorescence of CpyA-EGFP expressed in ΔavaB strain was far weaker than that in the wild-type strain, indicating that the protein was not correctly delivered to the vacuoles. In agreement with this observation, an increase in EGFP fluorescence was detected in the culture medium of ΔavaB strain, suggesting that the lack of avaB protein caused missorting of CpyA-EGFP (data not shown). These results, collectively, demonstrate that AvaB is required for the maintenance of normal vacuolar morphology and vacuolar protein sorting processes, but is not essential for the growth of A. nidulans on agar plates or in liquid media.
Homo- and heterooligomeric interactions of AvaB
It has been shown that S. cerevisiae Vam6p/Vps39p associates with the small GTPase Vam4p/Ypt7p and functions as a guanine nucleotide exchange factor, through which it promotes homotypic vacuolar membrane fusion and vacuolar biogenesis [16,18]. In addition, Caplan et al. have demonstrated that the human homolog of Vam6p/Vps39p, hVam6p, forms homooligomers, which is likely to be relevant to the in vivo function as a tethering factor for lysosome clustering and fusion . To gain insight into the molecular function of AvaB, the molecular interaction of AvaB with AvaA, a Vam4p/Ypt7p homolog in A. nidulans as well as homooligomer formation of AvaB were studied through yeast two-hybrid analysis. As shown in Fig. 5, both the heteromeric interaction between AvaA and AvaB and homomeric interaction of AvaB were observed, with the former apparently being more stable than the latter. The result indicates that AvaB assembles to heteromeric (with AvaA) as well as homomeric oligomers. Since in yeast only the interaction of Vam6p/Vps39p with the small GTPase Vam4p/Ypt7p has been reported, and for hVam6p the homooligomeric interaction, but not its association with small GTPase, has been reported, to our knowledge this is the first demonstration that a certain VAM6/VPS39 family protein displays the protein–protein interaction in both of these two ways.
In this paper we have described the molecular cloning of A. nidulans gene, avaB, which codes for the homolog of Vam6p/Vps39p of S. cerevisiae. The AvaB protein contains two putative domains, CNH and CLH, at the N-terminal and central regions, respectively, which have been proposed to be involved in the interaction with distinct target proteins. Indeed, the two-hybrid analysis using AvaB as a bait has demonstrated that it interacts with AvaA, a Vam4p/Ypt7p homolog in A. nidulans, suggesting that AvaB serves as a guanine nucleotide exchange factor for AvaA. Further support for this hypothesis has been obtained by the observation that the phenotype of ΔavaB strain is reminiscent of that of avaA-disrupted strain , suggesting that AvaB performs its function through the activation of AvaA GTPase. In addition, homooligomeric interaction of AvaB was also observed in the two-hybrid analysis. Although more direct experimental evidence is needed to unequivocally show that these putative domains in AvaB are functional motifs for protein–protein interaction, the results presented in this study suggest for the first time that a Vam6p/Vps39p family protein interacts in both ways.
The ΔavaB strain contained fragmented vacuoles and displayed retarded growth phenotype in various media conditions, in contrast to the prediction drawn from the observation by de Souza et al. that one of the cycloheximide-sensitive mutants (scy465) that contained fragmented vacuoles displayed fragility only to low pH and high osmolarity . Thus the loss of AvaB function causes not only the morphological alteration, but also the functional aberration of vacuoles that lead to the overall growth retardation in A. nidulans.
When the conidia formation was evaluated, we found that the loss of avaB resulted in significant reduction in the number of conidia compared to the wild-type (data not shown). This observation parallels with the previous study reporting that vam6/vps39 mutation had severe defects in spore formation in S. cerevisiae. Furthermore, the color of colony of ΔavaB mutant remained white-to-brown after 3 days of culture in MM medium, in contrast to the color of wild-type colony that changed from white to brown, and finally to green. These observations suggest that ΔavaB mutant has a defect in the differentiation process and the vacuolar function is required for the normal development of conidial structure. Further analysis on AvaB protein would help elucidate the physiological and developmental roles of fungal vacuoles.
This study was supported by a Grant-in-Aid for Scientific Research (B) (No. 12460041) to K.K. from the Ministry of Education, Science, Sports and Culture of Japan.
5-(and 6-)carboxy-2′,7′-dichlorofluorescein diacetate
enhanced green fluorescent protein