Mutations in Genes Encoding Sorting Nexins Alter Production of Intracellular and Extracellular Proteases in Aspergillus Nidulans

Mutations in Genes Encoding Sorting Nexins Alter Production of Intracellular and Extracellular Proteases in Aspergillus nidulans

Margaret E. Katz,*,1 Cara J. Evans,* Emma E. Heagney,*,2 Patricia A. vanKuyk,*,3 Joan M. Kelly† and Brian F. Cheetham* *Molecular and Cellular Biology, University of New England, Armidale, NSW 2351, Australia and †Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia Manuscript received August 18, 2008 Accepted for publication February 1, 2009

ABSTRACT XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17 homolog. Complementation of the sogA1 and sogB1 mutations and identification of nonsense mutations in the sogA2 and sogB1 alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17 mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG- independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

HE extracellular proteases of the filamentous fungus substitution (R186W) in the putative DNA-binding T Aspergillus nidulans can be used as a model to study domain of XprG. the response to starvation as production of extracellular XprG belongs to a newly identified group of proteins proteases is repressed in nutrient-sufficient growth that contains an Ndt80-like DNA-binding domain and medium and is stimulated by the absence of a carbon, belongs to the family of p53-like transcription factors nitrogen, or sulfur source, irrespective of whether protein (pfam.sanger.ac.uk/). Ndt80 activates the transcription is present (Cohen 1973a). The xprG gene plays a major of .150 genes during middle meiosis in Saccharomyces role in the regulation of extracellular proteases. Loss-of- cerevisiae (Chu et al. 1998). Nutrient limitation is re- function mutations in xprG abolish the production of quired for the initiation and completion of meiosis in S. extracellular proteases in response to carbon starvation cerevisiae (Esposito and Klapholz 1981). The ndt80 and the response to nitrogen and sulfur starvation is mutants arrest at the point in meiosis past which reduced (Katz et al. 2006). In addition, production of an progression through meiosis cannot be reversed by acid phosphatase in response to phosphate starvation is nutrient supplementation (Xu et al. 1995). Thus lost in xprG mutants indicating that XprG may be invol- Ndt80 may, like XprG, be involved in sensing nutrient ved in a general response to starvation (Katz et al. 2006). status (Katz et al. 2006). Mutations in xprG do not affect A single gain-of-function mutation in xprG has been meiosis. identified. Strains carrying the xprG1 mutation have Genetic evidence suggests that two noncatalytic increased extracellular protease activity in response to hexokinase-like proteins (HxkC and HxkD) are compo- carbon and, to a lesser extent, nitrogen starvation (Katz nents of the XprG regulatory pathway (Katz et al. 2000; et al. 1996). The xprG1 mutation results in an amino acid Bernardo et al. 2007). Loss-of-function mutations in hxkC and hxkD produce a similar phenotype to the xprG1 1Corresponding author: Molecular and Cellular Biology, University of gain-of-function mutation and are suppressed by xprG New England, Armidale, NSW 2351, Australia. mutations. HxkD is a nuclear protein whereas HxkC is E-mail: [email protected] associated with mitochondria (Bernardo et al. 2007). 2 Present address: Forensic DNA, Division of Analytical Laboratories, The binding of hexokinase to mitochondria blocks ICPMR, Lidcombe, NSW 2141, Australia. astorino 3Present address: Molecular Microbiology, IBL, Leiden University, NL- apoptosis in human tumor cells (P et al. 2002) 2300 RA Leiden, The Netherlands. and regulates programmed cell death in plants (Kim et al.

Genetics 181: 1239–1247 (April 2009) 1240 M. E. Katz et al.

TABLE 1 List of A. nidulans strains used in the study

Strain Genotypea Source MH2 biA1; niiA4 M. J. Hynes MH97 pabaA1 yA1 acuE215 M. J. Hynes MSF suA-adE20 yA1 adE20; acrA1; galA1, pyroA4; facA303; sB1; nicB8; riboB2 M. J. Hynes SA4B17 creA204 biA1; niiA4 M. J. Hynes SA15B12 biA1; creB15 M. J. Hynes SA27D8 biA1; creC27; niiA4 M. J. Hynes MK198 pabaA1; xprG2 prnD309; niiA4 Katz et al. (2000) MK307 pabaA1; xprG2 prnD309; niiA4 sogC1 This study MK308 pabaA1; sogA1; xprG2 prnD309; niiA4 This study MK309 pabaA1; sogA2; xprG2 prnD309; niiA4 This study MK310 pabaA1; xprG2 prnD309; sogB1; niiA4 This study MK314 suA-adE20/1 pabaA1/1 yA1/1 adE20/1; acrA1/1; galA1/1; pyroA4/1; This study facA303/1; sB1/1; xprG2/1 prnD309/1 nicB8/1; niiA4/1 riboB2/1 sogC1/1 MK315 suA-adE20/1 pabaA1/1 yA1/1 adE20/1; acrA1/1 sogA1/1; galA1/1; pyroA4/1; This study facA303/1; sB1/1; xprG2/1 prnD309/1 nicB8/1; niiA4/1 riboB2/1 MK316 suA-adE20/1 pabaA1/1 yA1/1 adE20/1; acrA2/1 sogA2/1; galA1/1; pyroA4/1; This study facA303/1; sB1/1; xprG2/1 prnD309/1 nicB8/1; niiA4/1 riboB2/1 MK317 suA-adE20/1 pabaA1/1 yA1/1 adE20/1; acrA1/1; galA1/1; pyroA4/1; This study facA303/1; sB1/1; xprG2/1 prnD309/1 sogB1/1 nicB8/1; niiA4/1 riboB2/1 MK362 acuE215; sogA1; riboB2 This study MK387 pabaA1/1 acuE215/1; sog A1/sogA2; prnD309/1 xprG2/1; niiA4/1 riboB2/1 This study MK414 pabaA1 yA2; argB2; xprGD1(xprGTargB)Katz et al. (2006) a For the meanings of gene symbols see Clutterbuck (1993).

2006). The similarity between XprG and Vib-1, which is cellular proteases in response to carbon starvation in an required for expression of genes during programmed xprG null mutant. cell death in Neurospora crassa (Dementhon et al. 2006), suggests that HxkC and XprG may have a role in reg- ulating programmed cell death as well as the response to nutrient depletion in A. nidulans. MATERIALS AND METHODS AreA is the transcriptional activator that mediates Aspergillus strains, media, growth conditions, genetic nitrogen metabolite repression in A. nidulans (Kudla techniques, and transformations: The Aspergillus nidulans et al. 1990). Though XprG is involved in controlling the strains used in this study are listed in Table 1. The media used atz production of extracellular proteases in response to for culture of A. nidulans was as described previously (K atz et al. 1996). The techniques used for genetic manipulation of nitrogen limitation, AreA function is also required (K A. nidulans have been described (Clutterbuck 1974). A. et al. 2006). The genetic data suggests that XprG may nidulans transformations were performed using the method modulate AreA-mediated activation of extracellular of Tilburn et al. (1983). protease gene expression (Katz et al. 2006). The aim Isolation of mutants: Conidial suspensions of strain MK198 of this study was to investigate whether a similar situation were irradiated with ultraviolet light (254 nm) as described previously (Katz et al. 1996). Mutagenized conidia were exists with respect to carbon regulation (i.e., Does XprG diluted and spread on minimal medium containing 1% skim modulate the activity of a transcription factor that milk as a carbon source to screen for revertants that produced mediates the response to carbon starvation?). The creA halos. gene encodes a transcriptional repressor, which controls Cloning of the sogA and sogB genes: To clone the sogA gene, carbon catabolite repression in A. nidulans (Dowzer nucleotides 154,070 to 156,746 (containing the A. nidulans elly homolog of VPS5) on contig 1.61 were amplified with Taq DNA and K 1991). Extracellular protease activity is in- polymerase using primers MK253 (59-GTAGAGATGCA creased in strains lacking CreA but the effect is XprG TAAGCTTGG-39) and MK256 (59-TCAAGAATTCACGAC dependent, suggesting that XprG acts downstream of TATGGAGCGAAG-39). The amplified DNA was digested with CreA (Katz et al. 2008). A genetic approach was then EcoRI and NsiI and inserted into pUC18 (Yanisch-Perron used to try to identify proteins that might be involved in et al. 1985). Five different vps5 clones were introduced into the pabaA1 sogA1 xprG2 strain (MK308) by cotransformation with the response to carbon starvation in partnership with the pabaA plasmid, pPABA-AT6 (R. B. Todd, unpublished XprG. We describe the isolation and characterization of results). Paba1 transformants were tested on medium con- suppressor mutations that restored production of extra- taining milk as a carbon source. Transformants that exhibited Sorting Nexins in A. nidulans 1241 a sogA1 xprG2 phenotype were analyzed by Southern blot analysis. To isolate the sogB gene, nucleotides 5832–9032 (containing the A. nidulans homolog of VPS17) on contig 1.36 were amplified with Taq DNA polymerase using primers MK285 (59-ACACTGCGCCATCGAGAATG-39) and MK286 (59-GATG GTTCAAATGCATACGC-39). The amplification product was introduced into the pabaA1 sogB1 xprG2 strain (MK310) by cotransformation and analyzed as described for the sogA gene. Nucleic acid extraction, blotting, and hybridization: RNA was prepared using the RNeasy Plant Mini kit according to the manufacturer’s instructions (QIAGEN). Northern blots were hybridized with 32P-labeled probes prepared using the Prime-a- Gene labeling system (Promega). Genomic DNA was extracted from A. nidulans by the method of Andrianopoulos and Hynes (1988). Southern blot hybridizations were performed using the DIG nonradioactive DNA labeling and detection system (Roche Applied Science). Protease enzyme assays: The enzyme assays used to measure protease activity in A. nidulans growth medium were performed as described previously (Katz et al. 1996). The results were expressed in arbitrary units (total absorbance units per gram dry weight of mycelium). Figure 1.—Phenotype of xprG2 revertants. Extracellular Gel electrophoresis and detection of protease activity: protease production of MH2 (WT), MK198 (xprG2), MK307 Nondenaturing polyacrylamide gel electrophoresis and de- (xprG2rev1), MK308 (xprG2rev2), MK309 (xprG2rev3), and tection of protease activity using a 1% agarose milk overlay was MK310 (xprG2rev4) on medium containing 1% skim milk performed as described by Katz et al. (1996). For the analysis as a sole source of carbon or nitrogen and growth on minimal of the effect of protease inhibitors, the gels were adjusted to medium containing 11 mm sodium molybdate is shown. The pH 5.3 after electrophoresis and then incubated at 37° in 0.15 m clear halo surrounding the colonies on medium containing sodium acetate buffer (pH 5.3) containing the inhibitor for 60 milk is due to extracellular protease activity. The full geno- min on a shaking platform prior to the application of the types of the strains are given in Table 1. agarose milk overlay. For detection of extracellular proteases, samples of filtered culture medium that contained 1 OD440 from the control strain, MH2, and an equivalent volume (adjusted for differ- in pseudorevertants was not due to a modification of the ences in mycelial dry weight) of medium from the mutant pH of the culture medium. strains were prepared as described by Katz et al. (2008). For The four pseudorevertants displayed similar altered detection of intracellular proteases, powdered lyophilized colony morphology, compact growth, and reduced con- mycelium was suspended in H2O at a concentration of 10 idiation. Although the pseudorevertants produced halos mg/ml and frozen. After thawing, the mycelial debris was removed by centrifugation. An equal volume of sample buffer on media containing milk as a carbon or nitrogen source, (40% glycerol 0.3 m Tris–HCL pH 6.8) and 5 ml 0.1% the ability of the pseudorevertants to utilize protein (1% bromophenol blue were added to duplicate 25-ml aliquots BSA or 1% casein) as a carbon or nitrogen source was not and were loaded in adjacent wells of the gel. significantly increased. Outcrosses showed that each pseudorevertant carried an extragenic suppressor of the xprG2 mutation. The altered colony morphology, RESULTS compact growth, and reduced conidiation of the segre- Isolation and genetic characterization of xprG2 gants carrying the suppressor mutations in xprG1 and pseudorevertants: The xprG2 allele contains a frame- xprG2 genetic backgrounds were similar. shift mutation in the ninth codon of the predicted open Diploids (MK314–MK317) constructed from the four reading frame. To identify suppressors of the xprG2 pseudorevertants and the mapping strain MSF for mutation, conidia from an xprG2 strain (MK198) were haploidization analyses exhibited normal colony mor- irradiated with ultraviolet light and spread on medium phology indicating that the mutations were recessive. The containing milk as a carbon source. Four pseudorever- suppressor mutation in xprG2rev3 was located on chro- tants (MK307–MK310) that produced halos were iso- mosome II and the mutation in xprG2rev4 was located on lated (Figure 1). Protease enzyme assays showed that the chromosome VIII. The mutation in xprG2rev2 was due to response to carbon starvation was partially restored but a translocation involving chromosomes II and VII and there was no difference in extracellular protease levels xprG2rev1 carried a rearrangement involving three chro- in response to nitrogen or sulfur starvation in the four mosomes (I, VI, and VIII). pseudorevertants, when compared to the xprG2 strain The chromosome II translocation breakpoint in (MK198) from which they were derived (Figure 2). The xprG2rev2 and the suppressor mutation in xprG2rev3 final pH of the carbon-free culture medium was the were both mapped to within 5 MU of creB. Phenotypic same (pH 6.4) for xprG1, xprG2, and pseudorevertant analysis of a diploid (MK387), constructed to test for strains. Thus, the increase in protease activity detected complementation, showed pseudorevertants 2 and 3 1242 M. E. Katz et al.

Figure 2.—Extracellular protease activity in xprG2 revertants. Protease activity (in arbitrary units) was measured in ﬁltered culture medium from strains MH2 (WT), MK198 (xprG2), MK307 (xprG2rev1), MK308 (xprG2rev2), MK309 (xprG2rev3), and MK310 (xprG2rev4) exposed to four indicated different nutrient conditions. The results are the average for three cultures and the standard deviations are shown. The protease enzyme assays were performed as described in materials and methods.

carry allelic suppressor mutations, designated sogA1 was determined. A nonsense mutation was identified in (suppressor of xprG) and sogA2, respectively. The codon 383 (supplemental Figure S1). xprG2rev4 mutation (designated sogB1) was mapped to Identification of the sogB gene: In S. cerevisiae Vps5p 2.1 MU from methE and 10 MU from gatA on chromo- forms a complex with Vps17p (Horazdovsky et al. some VII. The chromosomal rearrangement in xprG2 1997). An A. nidulans homolog of VPS17 (AN2224) is on pseudorevertant 1 involved chromosomes I, VI, and VIII, whereas sogA was located on chromosome II and sogB on chromosome VII. Thus the suppressor mutation in xprG2rev1 was located in a third gene, sogC. Identification of the sogA gene: The genetic mapping data and colony morphology of the xprG2 pseudorevertants suggested that sogA1 and sogA2 might be alleles of acrB, which is also linked to creB (Boase et al. 2003). Three of the four pseudorevertants exhibited increased sensitivity to molybdate similar to acrB mutants (Figure 1). However, transformation experiments indicated that acrB did not complement the sogA1 mutation. To confirm that sogA1 was not an allele of acrB, p4AcrB was used to probe Southern blots of genomic DNA from sogA1 and sogA1 strains. The results showed that p4AcrB hybridized to fragments that contained the sogA1 translocation breakpoint but that the translocation breakpoint was located in AN3594, rather than in acrB (Figure 3, data not shown). AN3594 is a homolog of the S. cerevisiae VPS5 gene, which encodes a sorting nexin required for the separation of secreted and vacuolar proteins in the late Golgi (Nothwehr and Hindes 1997). To confirm that AN3594 was sogA, clones containing AN3594 were used in cotransformation experiments with xprG2rev2 (MK308) to test whether AN3594 could complement the sogA1 mutation. A plasmid containing the pabaA gene, pPABA-AT6 (R. B. Todd, Figure 3.—Localization of the sogA and sogB genes. (A) A unpublished results), was used as a selectable marker. Southern blot of genomic DNA from strains MH97 (WT) and 1 MK308 (sogA1) probed with a plasmid containing the acrB Of 23 paba transformants, three displayed a phenotype oase 1 gene (B et al. 2003) is shown. The restriction map below indistinguishable from the sogA xprG2 strain MK198 the blots shows the predicted ORFs in the region surrounding (Figure 4). Southern blot analysis confirmed that these the acrB gene in the A. nidulans genome database (www.broad. threetransformantscarriedthe AN3594 plasmids whereas mit.edu/annotation/fungi/aspergillus/index.html). The po- three paba1 transformants that resembled xprG2rev2 did sition of the sogA1 translocation breakpoint is indicated. not contain the plasmids (data not shown). (B) The genetic and physical map of the region surrounding the methE gene on chromosome VII is shown. The position of To confirm that xprG2rev3 also carries a mutation in a VPS17 homolog corresponds to the position of sogB, which the VPS5 homolog, AN3594, the coding region of the genetic mapping showed was located 2.1 MU from methE and gene was amplified from MK309 and its DNA sequence 10 MU from gatA. Sorting Nexins in A. nidulans 1243

Figure 5.—Levels of the prtA transcript in the xprG2 revertants. Northern blots were prepared using total RNA extracted from strains MH2 (WT), MK198 (xprG2), MK307 (xprG2rev1), MK308 (xprG2rev2), MK309 (xprG2rev3), and Figure 4.—Complementation of the sogA1 and sogB1 mu- MK310 (xprG2rev4) after transfer to media lacking a carbon tations. (Top plates) The phenotype of sogA1 transformants, (left blot) or nitrogen source (right blot) as indicated. RNA the transformation recipient strain MK308 (sogA1 xprG2), extracted from strain MH2 (WT) after growth in nutrient- MH2 (WT), and MK198 (xprG2) on medium containing 1% sufficient medium is in the left lane of each blot. The blots skim milk as a sole source of carbon or nitrogen is shown. were probed with 32P-labeled pBC174 plasmid containing Transformants were generated by cotransformation of a sogA1 the entire coding region of the prtA gene (Katz et al. xprG2 pabaA2 strain (MK308) with a plasmid containing the 1994). An identical ethidium bromide stained gel of the VPS5 homolog (AN3594) and a plasmid containing the pabaA RNA used in each blot is shown in the bottom panels. gene, pPABA-AT6 (R. B. Todd, unpublished results). Three paba1 sog1 transformants (sogA1 transformants), which contain the VPS5 plasmid and show an identical phenotype to and 49% identity in the 115 amino acid PX (phosphoi- the xprG2 strain, are shown in the middle row. Three paba1 nositide-binding) domain (http://pfam.sanger.ac.uk/ transformants (sogA transformants) that did not display a family?acc¼PF00787). In contrast, there is no difference sogA1 phenotype and do not contain the VPS5 plasmid are in the overall similarity of the 574-amino-acid SogB shown in the bottom row. (Bottom plates) The phenotype of sogB1 transformants, the transformation recipient strain sequence to Vps17p (38% identity) and the PX domain MK 310 (sogB1 xprG2), MH2 (WT), and MK198 (xprG2)on (35% identity). The nonsense mutations in sogA2 (co- medium containing 1% skim milk as a sole source of carbon don 383) and sogB1 (codon 262) are downstream from or nitrogen is shown. Transformants were generated by co- the sequences encoding the PX domains of SogA transformation of a sogB1 xprG2 pabaA2 strain (MK310) with (codons 161–275) and SogB (codons 115–229). Thus, it a PCR product carrying the VPS17 homolog (AN2224) and a plasmid containing the pabaA gene, pPABA-AT6 (R. B. Todd, is possible that sogA2 and sogB1 are not null mutations. unpublished results). Five paba1 sog1 transformants (sogB1 The translocation breakpoint in sogA1 is between transformants) that show an identical phenotype to the xprG2 codons 246 and 294 and, therefore, may also not disrupt strain, are shown in the middle row. Two paba1 transformants 1 the PX domain. (sogB transformants) that did not display a sogB phenotype Within the S. cerevisiae VPS5 gene there is a gene are shown in the bottom row. (VAM10) on the complementary strand that is required for vacuole morphogenesis (Kato and Wickner 2003). chromosome VII in the same position that genetic No open reading frame encoding a protein similar to mapping data indicated as the location of sogB (Figure Vam10p was found in sogA. 3). A similar strategy to that employed for sogA was used Expression of the serine protease gene prtA in the to confirm that AN2224 could complement the sogB1 revertants: Loss-of-function mutations in xprG abolish mutation (Figure 4). production of extracellular proteases in response to The coding region of AN2224 gene was amplified carbon starvation. Northern blot analysis was performed from MK310 to identify the sogB1 mutation. Sequence to determine whether extracellular protease gene ex- analysis revealed the presence of a nonsense mutation pression is restored in the four revertants (Figure 5). in codon 262. The blots were probed with the prtA gene, which Molecular characterization of sogA and sogB: The encodes an extracellular serine protease that is respon- intron/exon structure of the AN3594 (sogA) and AN2224 sible for most of the extracellular protease activity (sogB) coding regions were confirmed by RT–PCR and detected in A. nidulans (Katz et al. 1994; vanKuyk DNA sequence analysis. The annotation of AN2224 et al. 2000). In the xprG1 [wild-type (WT)] strain high proved to be correct (www.broad.mit.edu/annotation/ levels of prtA transcript were produced during carbon fungi/aspergillus/index.html) but the second intron in starvation but no transcript was detected in the xprG2 AN3594 was in the wrong position (supplemental Figure mutant. In the four revertant strains, the prtA transcript S1). The corrected 567-amino-acid SogA sequence was detected but the level was much lower than in the shows 27% amino acid identity with S. cerevisiae Vps5p wild-type strain. In the northern blot of RNA produced 1244 M. E. Katz et al.

TABLE 2 The effect of protease inhibitors on the activity of proteases in A. nidulans

Bands of protease activityb Inhibitora 123456 10 mm EDTA 11111 10 mm 1,10-o-phenanthroline 11111 1% DTT 11111 2mm PMSF 1666 6 2 mg/ml aprotinin 1666 50 mg/ml trypsin inhibitor 1 66 10 mm PCMPS 1 66 8 mm leupeptin 16666 570 mm TLCK 11111 1 mm pepstatin A 111111 Figure 6.—Detection of protease activity in nondenaturing a polyacrylamide gels. (A) Protease activity in concentrated sam- Protein gels were incubated in each inhibitor for 1 hr ples of filtered, carbon-free culture medium (E) and mycelial prior to protease detection as described in materials and extract (I) from strain MH2 was detected using a milk agarose methods. b overlay as described in materials and methods. For the The bands are numbered as in Figure 6. The activity in detection of intracellular proteases (I), mycelium grown in each band was compared to a control that was not exposed nutrient-sufficient medium was used. For detection of extracel- to inhibitor prior to protease detection and the effect of lular proteases (E), the medium lacked a carbon source. Pro- the inhibitor was scored as total loss of activity (), partial in- teolytic bands are labeled 1–6 in order of increasing mobility. hibition of protease activity (6) or no inhibition (1). Band 5 has been shown to be the product of the prtA gene (van- Kuyk et al. 2000). (B) Protease activity in filtered carbon-free culture medium (top) and mycelia (bottom) from strains profiles differ from each other as well as from bands 2, MK198 (xprG2), MK307 (xprG2rev1), MK308 (xprG2rev2), MK309 (xprG2rev3), MK310 (xprG2rev4), and MH2 (WT). 3, and 4. The identity of band 5 is known. Gene disruption experiments have shown that it is the product of the prtA gene (vanKuyk et al. 2000). None of the during nitrogen starvation, high levels of the prtA bands were affected by the aspartic protease inhibitor transcript were observed in the wild-type strain and a pepstatin A. faint band was detected in the xprG2 mutant. None of Analysis of the spectrum of proteases in the filtered the xprG2 revertants showed higher levels of prtA culture medium of the xprG2 revertants showed very transcript than the xprG2 mutant indicating that the high levels of the three intracellular protease bands sog mutations do not restore the response to nitrogen (bands 2, 3, and 4 in Figure 6B, top). These results starvation. suggest that proteases that are normally found in the Secretion of intracellular proteases: Mutations in the vacuole are secreted in the revertants. A band corre- S. cerevisiae VPS5 gene result in secretion of an intracel- sponding to PrtA was detected in the xprG2 sogC1 strain lular protease that is normally located within the vacuole (xprG2 revertant 1) but not in the other three revertants. (Robinson et al. 1988). The proteases found in the No bands with protease activity were visible in the xprG2 culture medium of an A. nidulans xprG1 strain (MH2) mutant. High levels of the activity corresponding to during carbon starvation were characterized using pro- intracellular protease bands 2, 3, and 4 were also tease inhibitors and nondenaturing gel electrophoresis. detected in mycelia (Figure 6B, bottom), indicating that Six bands of proteolytic activity were observed in culture abnormal secretion of this protease has not depleted the filtrate from carbon starvation conditions (Figure 6A, intracellular pool. lane E). Metal chelating agents (EDTA, 10-o-phenanthroline and DTT) inhibit the proteolytic activity of Band 1 indicating that this band results from the activity DISCUSSION of a metalloprotease (Table 2). Bands 2, 3, and 4 have identical susceptibility to serine protease inhibitors In eukaryotes, a series of membrane-enclosed com- (aprotinin, leupeptin, PCMPS, PMSF, TLCK, and trypsin partments are involved in transport of proteins to the inhibitor), indicating that they are all forms of the same cell surface and, in the reverse direction, transport of serine protease. These bands were also observed in cell surface proteins marked for degradation. In the late extracts of mycelia grown in glucose and are likely to Golgi, newly synthesized proteins destined for the cell be due to the activity of an intracellular serine protease surface or secretion (e.g., extracellular proteases) are (Figure 6A, lane I). Bands 5 and 6 also show susceptibility sorted from vacuole-bound proteins (e.g., intracellular to serine protease inhibitors but their susceptibility proteases), which move into the endosomal system. Cell- Sorting Nexins in A. nidulans 1245 surface proteins targeted by ubiquitination for degra- 2004). SNX1 has recently been shown to be involved in dation in the vacuole, also enter the endosomal system retromer-independent transport of a G protein-coupled from whence they can be retrieved by retrograde trans- receptor to the lysosome for degradation (Gullapalli port to the Golgi. In addition to cell-surface proteins, et al. 2006). Both SNX1 and SNX2 associate with a variety other proteins that are known to be subject to retrograde of cell surface receptors in vivo and in vitro (Haft et al. transport from endosomes to the Golgi in S. cerevisiae 1998; Heydorn et al. 2004). SogA and SogB may also be include the acid hydrolase receptor Vps10p, the pepti- involved in the degradation of cell surface proteins in A. dases Kex2p and Ste13p, and the v-Snare Snc1p (re- nidulans. Mutations in both these genes increase sensi- viewed in Bonifacino and Rojas 2006). tivity to molybdate. One mechanism by which this might Two of the genes identified in this study are putative occur is through an increase in the uptake of molybdate homologs of the S. cerevisiae vacuolar protein-sorting through decreased degradation of a molybdate trans- (VPS) genes, VPS5 and VPS17, which are involved in porter in the mutants. retrograde transport from endosomes to the Golgi Northern blot analysis showed that transcript from (Horazdovsky et al. 1997). In S. cerevisaie, vps5 and the extracellular serine protease gene prtA was detected vps17 mutants contain fragmented vacuoles and are (albeit at low levels) in the xprG2 revertants. PrtA activity defective in the sorting and processing of vacuolar was also detected in one of the four revertants using hydrolases including carboxypeptidase Y, proteinase A, protease zymograms. This finding suggests that extra- and proteinase B (Banta et al. 1988; Robinson et al. cellular protease gene expression is activated during 1988). Enzymes that are destined for the vacuole are carbon starvation by an xprG-independent mechanism separated from secreted proteins by binding to a sorting in the revertants. The pathways involving creA, creB, and receptor,Vps10p, in a late Golgi compartment (Vidaet al. creC are unlikely to be responsible as mutations in these 1993; Marcusson et al. 1994). The vacuolar proteins are genes do not suppress the xprGD1 null mutation (Katz then delivered to a prevacuolar endosome. Once its et al. 2008). cargo is delivered, Vps10p is returned by the retromer The properties of the extracellular and intracellular complex back to the Golgi where it is then reused proteases that were detected in this study are similar to (Seaman et al. 1998). The recycling of Vps10p is essential those reported by Cohen (1973b). Cohen identified, in for the delivery of vacuolar enzymes and this recycling the culture filtrate of A. nidulans, four proteases (a, b, g, depends on Vps5p and Vps17p, which are components and e) that were distinguished on the basis of electro- of the retromer complex (Horazdovsky et al. 1997). phoretic mobility, inhibitor sensitivity, substrate prefer- Vps26p, Vps29p, and Vps35p are also components of the ence, and pH optimum. Extracellular proteases g and e retromer (Bonifacino and Rojas 2006). In vps5 and were susceptible to serine protease inhibitors and are vps17 mutants, Vps10p is not present in the late Golgi similar to bands 5 and 6 in this study. Band 5 has been where protein sorting normally occurs and, thus, car- shown to be the prtA gene product (Katz et al. 1994; boxypeptidase Y is secreted rather than targeted to the vanKuyk et al. 2000). Extracellular protease a was not vacuole. inhibited by any of the protease inhibitors tested by The data presented here suggest the A. nidulans vps5 Cohen (1973b) but the mobility of a was altered by (sogA) and vps17 (sogB) homologs perform a similar role EDTA (Cohen 1973b). The protease activity in band 1, and that mutations in these genes result in the secretion which is sensitive to metal-chelating agents and thus is a of proteases that are normally intracellular. The sogC metallo-protease, probably corresponds to protease a. gene has not yet been isolated due to the complex The fact that Cohen (1973b) treated his samples with nature of the sogC1 mutation. However, a high level of protease inhibitors prior to electrophoresis rather than intracellular protease was also detected in the culture afterwards as in this study and that protease inhibition medium of the sogC1 mutant, which indicates that sogC by EDTA is reversible may account for the differences in is also likely to encode a Vps homolog. The region the two studies. adjacent to the sogA gene contains a second sorting Like Cohen (1973b), who found three forms of an nexin (AN3584) and appears to contain a cluster of intracellular serine protease (b), three bands (2, 3, and other genes involved in protein trafficking and degra- 4) of protease activity with identical inhibition profiles dation (supplemental Table S1). were found in mycelial extracts in this study. This Mammalian genomes encode two homologs of Vps5p protease activity was present at very high levels in the (sorting nexins SNX1 and SNX2) but no Vps17p culture medium and mycelia of the xprG2 revertants. It is homolog (Kurten et al. 1996; Haft et al. 1998, 2000). not known whether there are higher levels of the The evidence suggests that mammalian sorting nexins, intracellular protease during carbon starvation in A. like Vps5p, are components of the mammalian retromer nidulans but in A. niger, a potential homolog of the gene (Arighi et al. 2004; Seaman 2004). Sorting nexins are encoding bands 2–4 (pepC) is expressed constitutively also involved in the degradation of the epidermal growth (Jarai et al. 1994). It remains to be determined whether factor receptor, though the mechanism by which this the secretion of intracellular protease triggers increased occurs is not clear (Carlton et al. 2004; Gullapalli et al. production or whether the sog mutations activate a 1246 M. E. Katz et al. signaling pathway (e.g., a stress response pathway) that Haft, C. R., M. de la Luz Sierra,R.Bafford,M.A.Lesniak,V.A. arr leads to increased expression by some other mechanism. 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