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Table of Contents
Abstract
Keywords
Introduction
Results And Discussion
Acknowledgments
Author Contributions
Declaration Of Interests
Materials And Methods
Plasmids
AID* System
Fluorescence Microscopy
Colony Dot Blot
Mammalian Cell Methods
Statistical Analysis
Abstract
The vacuolar H+-translocating ATPase (V-ATPase) is the major proton pump for intraorganellar acidification. Therefore, the integrity of the V-ATPase is closely associated with cellular homeostasis, and mutations in genes encoding V-ATPase components and assembly factors have been reported in certain types of diseases. For instance, the recurrent mutations of ATP6AP1, a gene encoding a V-ATPase accessory protein, have been associated with cancers and immunodeficiency. With the aim of studying V-ATPase-related mutations using the yeast model system, we report that Big1 is another homolog of ATP6AP1 in yeast cells, and we characterize the role of Big1 in maintaining a fully functional V-ATPase. In addition to its role in acidifying the vacuole or lysosome, our data support the concept that the V-ATPase may function as part of a signaling pathway to regulate macroautophagy/autophagy through a mechanism that is independent from Tor/MTOR.
The vacuolar H+-translocating ATPase (V-ATPase) is the major proton pump for intra- organellar acidification. Therefore, the integrity of the V-ATPase is closely associated with cellular homeostasis, and mutations in genes encoding V-ATPase components and assembly factors have been reported in certain types of diseases. For instance, the recurrent mutations of ATP6AP1, a gene encoding a V-ATPase accessory protein, have been associated with cancers and immunodeficiency. With the aim of studying V-ATPase-related mutations using the yeast model system, we report that Big1 is another homolog of ATP6AP1 in yeast cells, and we characterize the role of Big1 in maintaining a fully functional V-ATPase. In addition to its role in acidifying the vacuole or lysosome, our data support the concept that the V-ATPase may function as part of a signaling pathway to regulate macroautophagy/autophagy through a mechanism that is independent from Tor/MTOR.
Keywords
Autophagy, lysosome, stress, vacuole, V-ATPase
Introduction
The vacuolar H + -translocating ATPase (V-ATPase) is a conserved and ubiquitously expressed protein complex, which is the main regulator of intra-organellar acidification. The VATPase is a multi-subunit complex composed of two large domains, the cytosolic V1 domain, which hydrolyzes ATP, and the membrane-embedded V0 domain, which transports protons. The V0 and V1 domains can assemble separately and the assembly of the two domains is highly regulated [1]. In yeast cells, the assembly of the V0 domain components occurs in the endoplasmic reticulum (ER) membrane and depends on several assembly factors residing in the ER, including integral membrane protein Vma12 and Vma21 and the peripheral membrane protein Vma22 [2-6]. In addition, Voa1 has also been identified to participate in the assembly of the V0 domain [7]. The assembled V0 domain is transported to the Golgi membrane where the V1 domain assembles with it [6]. Even though V-ATPase assembly in mammalian cells is less well studied than in yeast cells, homologs of most of the yeast V-ATPase assembly factors have been identified and several studies resolving mammalian V-ATPase structure have shed light on VATPase assembly in more complex eukaryotes [8, 9]. As the main proton pump responsible for the acidification of endomembrane compartments, including the lysosome and vacuole, the V-ATPase plays a critical role in autophagy, a conserved degradation pathway depending on the vacuole/lysosome; V-ATPasemediated vacuolar/lysosomal acidification affects the fusion of the autophagosome (the doublemembrane vesicle that transports autophagic cargo) and the vacuole/lysosome [10], and the dysregulation of vacuolar/lysosomal pH can result in the disrupted lysosomal degradation of autophagic cargos [11]. In addition, the V-ATPase also plays a significant role in other types of autophagy, such as xenophagy and non-canonical autophagy, independent from its major function in mediating acidification [12-14]. More importantly, mutations in, and the dysregulation of, the V-ATPase have been linked with multiple human diseases, such as cancer, neurodegenerative disorders, and infectious diseases [1, 15]. Various genes encoding V-ATPase structural components and assembly factors have been demonstrated to be highly mutated in follicular lymphoma (FL), including VMA21 and ATP6V1B2, and some mutations cause abnormal autophagy [16, 17]. ATP6AP1/Ac45, one of the V-ATPase accessory proteins in the mammalian system, is also frequently mutated in FL cases [16, 18]. In addition, ATP6AP1 is highly mutated in other cancers and diseases, such as granular cell tumors and immunodeficiency [19, 20], suggesting the importance of this gene. Given the close connection between the V-ATPase and autophagy, it is possible that ATP6AP1 functions as an autophagy regulator and that these mutations may result in an alteration in autophagy activity, thereby contributing to disease development or progression. Thus, a better understanding of mutations in genes encoding the V-ATPase and/or its assembly factors may provide useful insights for designing therapeutic approaches. As the V-ATPase is essential for eukaryotic cell viability except in fungi [7] and the vacuole, the equivalent organelle of the lysosome, is relatively large, yeast cells serve as a good model organism to study the V-ATPase. Therefore, we decided to examine the role of ATP6AP1 in autophagy by studying its homolog in yeast cells. In this study, we determined that Big1, a largely uncharacterized protein, is a second homolog of ATP6AP1 and is important for a fully functional V-ATPase in yeast cells. In addition, we found that the removal of Big1 results in autophagy induction. Similarly, the knockdown of ATP6AP1 in human cells also causes increased autophagy, suggesting that the V-ATPase may function as a signal in regulating bulk autophagy besides its role in acidifying the lysosome or vacuole.
Results and discussion
Yeast Big1 has a function similar to mammalian ATP6AP1 Using HHpred, a server for protein homology detection and structure prediction (https://toolkit.tuebingen.mpg.de/tools/hhpred) [21], we determined that the sequence of the yeast protein Big1 has significant similarity to the N terminus of ATP6AP1 (E = 3.8e-22) (Figure 1A and S1). In addition, Voa1 has also been shown to have significant similarity to both the N and C termini of ATP6AP1 (E = 0.045 and 3.4e-9, respectively). Compared with Voa1, which has been defined as a V-ATPase assembly factor with known structure and localization, very few studies have focused on Big1. The current available information concerning Big1 is that it is located in the ER and is required for the synthesis of -l,6-glucan, an essential polymer involved in the cell wall attachment of many surface mannoproteins [22]. We examined the localization of Big1 tagged with green fluorescent protein (GFP) relative to the ER marker Sec63-RFP and found substantial overlap (Figure 1B), confirming that Big1 resides in the ER. Given the similarity between sequences of Big1 and ATP6AP1, we suspected that Big1 may also function in V-ATPase assembly. Yeast cells with deficient V-ATPase activity are viable but exhibit a Vma - phenotype, characterized by the inability to grow in medium buffered to pH 7.5 and/or in the presence of a higher extracellular concentration of calcium or heavy metals [23]; culture medium at pH 5.0 suppresses the growth defect. Vma21 plays a major role in VATPase assembly [2]. For example, the phenotype associated with the voa1 deletion is strongly exacerbated in cells with partially defective Vma21 [7]. Vma21 requires a C-terminal dilysine motif to be retained in the ER; when the dilysine is mutated to diglutamine (Vma21[QQ]), Vma21 is mislocalized resulting in reduced V-ATPase assembly and activity [7]. Accordingly, we generated big1∆ cells in a Vma21[QQ] background. Compared with the wild-type cells, big1 vma21[QQ] cells showed a growth deficiency on a YPD plate buffered to pH 7.5 in the presence of 35 mM calcium. In contrast, there was no significant difference in growth on YPD buffered to pH 5.0. The growth phenotype was partially rescued by adding back Big1 or, to a lesser extent, human ATP6AP1 driven by the yeast ZEO1 promoter (Figure 1C). Expressing the N terminus of ATP6AP1, corresponding to the region that is homologous to Big1, recues the growth phenotype better than full-length ATP6AP1, which may be due to inefficient Kex2/furin cleavage in yeast cells, an important step for the full function of ATP6AP1 [24] (Figure 1C). This finding suggests that Big1 is one of the functional homologs of ATP6AP1 and may function in V-ATPase assembly in yeast. Big1 depletion leads to less V-ATPase on the vacuole and deficient enzyme activity Some studies classify BIG1 as an essential gene [22, 25], and big1 cells grow slowly (doubling time being approximately 2 h). Thus, to avoid potential indirect effects brought about by the deletion of BIG1, we took advantage of the auxin-inducible degron (AID*) system to conditionally knock down BIG1 expression. This system is well established in yeast cells to temporally control gene expression by mediating proteasomal degradation of targeted proteins in an auxin-dependent manner [26]. Therefore, these cells can be maintained as essentially wild- type for normal growth and Big1 can be removed in a temporal manner. The phenotype of VATPase assembly defects is cumulative, and only newly synthesized complexes are affected; thus, some time is necessary for the phenotype to be strong enough to be detected. Accordingly, we treated the Big1-AID* cells with 300 M IAA for 12 h before collecting samples, which is enough for essentially complete loss of detectable Big1 (Figure 2A). To directly determine the impact on V-ATPase assembly after the knockdown of BIG1, yeast vacuoles were isolated, and the protein levels of some V-ATPase components were detected by western blot (Figure 2B). Compared with the control cells treated with dimethyl sulfoxide (DMSO), we observed that vacuolar protein levels of Vph1, a V0 domain component, and Vma2 and Vma8, two V1 domain components, significantly decreased in the cells treated with IAA. Vph1 levels were also decreased in the total cell extract, which fits with the observation that this subunit is unstable when V-ATPase assembly is defective [7]. In addition, using isolated vacuoles, V-ATPase activity was measured in a bafilomycinA1 (BafA1)-dependent manner. Consistent with the protein level, the V-ATPase activity was significantly reduced in the Big1-AID* cells treated with IAA relative to the control group (Figure 2C). These results indicate that the removal of Big1 leads to a reduced level of V-ATPase subunits on the vacuole, suggesting that Big1 is critical in maintaining the normal level and function of the V-ATPase. We further determined whether Big1 interacts with V-ATPase. We used the bimolecular fluorescence complementation (BiFC) assay, which is based on the association between fragments of a fluorescent protein when they are tethered in the same macromolecular complex. In this assay, two fragments, VN and VC of Venus, a yellow fluorescent protein, are fused to two proteins of interest. The interaction between the proteins facilitates the association between the VN and VC chimeras to produce a bimolecular fluorescent complex [27]. We tagged the VN fragment to Big1 (Big1-VN) and VC to the V-ATPase component Vph1 (VC-Vph1), and assembly factors, including Vma21 (Vma21-VC) and Voa1 (Voa1-VC) and imaged cells by fluorescence microscopy. Clear YFP puncta were observed in the Big1-VN cells expressing VCVph1 or Vma21-VC (Figure 2D). Even though the puncta were not as bright, they were also detected in the Big1-VN cells expressing Voa1-VC (Figure 2D). The faint puncta in these strains could be explained by the localization of Voa1 in the middle of the V-ATPase Vma3/c-ring [28], which make the interaction or colocalization hard to detect by the BiFC assay. The interaction between Big1 with V-ATPase components and assembly factors is similar to Vma21, which can interact with Vph1 and to a lesser extent Voa1 (Figure S2A)[4]. We further examined the site of these interactions and found that most of those between Big1 and Vph1 occurred at the ERvacuole contact site, whereas more of the interactions between Vma21 and Voa1 were on the ER (Figure S2B). In addition, the interaction between Big1 and Vma21 or Vph1 could be detected by co-immunoprecipitation (Figure S2C and D). These results demonstrate that Big1 interacts with some the V-ATPase components and assembly factors and may function in facilitating VATPase assembly though these interactions. Big1 depletion induces autophagy Given the close association between V-ATPase and autophagy, it is possible that Big1 could be an autophagy regulator in yeast cells. Using the Big1-AID* strain, we decided to investigate the impact on autophagy brought about by the reduced expression level of BIG1. After treating the Big1-AID* cells with IAA for 12 h under nutrient-rich conditions, the cells were transferred to nitrogen-starvation medium (SD-N) and IAA treatment was continued in this condition to maintain the BIG1 expression at a minimal level. We utilized the GFP-Atg8 processing assay, where Atg8 is tagged with GFP at its N terminus, to evaluate autophagy flux. The principle of this assay is that the GFP-Atg8 on the inner membrane of the autophagosome will be delivered to the vacuole after the autophagosome fuses with this compartment. Within the vacuole, Atg8 will be degraded by vacuolar hydrolases, but GFP is more resistant to degradation. Therefore, free GFP is generated and the conversion of GFP-Atg8 to free GFP reflects autophagy flux. After the removal of Big1 by treating the cell with IAA for 12 h, we noticed the GFP-Atg8 cleavage even before starvation (Figure 3A; 0 min, +IAA), indicating that autophagy was activated by the reduced Big1 protein level under nutrient-rich conditions; autophagy is normally kept at a low basal level until cells are stressed. This induced autophagy can be suppressed by adding back another copy of Big1 from the plasmid (Figure S3A), indicating that this phenotype resulted solely from the removal of Big1. To provide further support for the induction of autophagy under nutrient-rich conditions, we measured the expression level of some ATG (autophagy related) genes. We chose ATG1, ATG7, ATG8, ATG9, and ATG41, which are all essential for the efficient activation of autophagy, because each of these genes shows an increase in expression upon autophagy induction and the level of Atg7, Atg8 and Atg9 clearly correlates with autophagy activity [29, 30]. The expression of each of these genes increased after IAA treatment compared with the control DMSO-treated cells in the nutrient-rich medium (Figure S3B). This result confirmed the induction of autophagy by the removal of Big1 protein under nutrient-rich conditions. After starvation, the autophagy activity also showed an increase in the IAA treatment group, however, to a lesser extent compared with the cells in growing conditions (Figure 3A and B, 45 and 90 min). This finding fits with the general observation that it is easier to detect positive effects of mutations on autophagy under growing conditions when autophagy is typically downregulated [31]; under starvation conditions, autophagy is highly upregulated due to the inhibition of various negative factors, making it difficult to detect the relatively smaller increase in activity such as can be seen in nutrient-rich conditions. In addition, when we followed the autophagy induction across the 12-h IAA treatment, we found that the GFP-Atg8 cleavage under growing conditions occurred after 4-h IAA treatment and a peak occurred at approximately 8 h (Figure S3C). These results suggest that the removal of Big1 induces autophagy and that the primary impact is seen under nutrient-rich conditions. One reason that autophagy is maintained at a low basal level when nutrients are replete is due to the activation of Tor, a key nutrient sensor and the primary negative regulator of autophagy in yeast cells. Activated Tor phosphorylates various Atg proteins including Atg1 and Atg13 [32] and inhibits the formation of the principle autophagy initiating complex consisting of Atg13, Atg1 and Atg17-Atg31-Atg29 [33, 34]. Because we saw the abnormal activation of autophagy under nutrient-rich conditions after knocking down the expression of BIG1, we next analyzed the Tor activation status by analyzing the phosphorylation of Atg13, Gln3 and Npr1, which are phosphorylated in a Tor-dependent manner [35-37]. Accordingly, we prepared protein extracts and analyzed them by SDS-polyacrylamide gel electrophoresis (PAGE) and western blot; both of these proteins migrate at a higher molecular mass when phosphorylated. Under nutrient-rich conditions, Atg13, Gln3 and Npr1 migrated at a higher position, suggesting the phosphorylation of these two proteins in Big1-AID* cells treated with either DMSO or IAA; in contrast, there was an increase in mobility on the gel when cells were starved, corresponding to a decrease in phosphorylation (Figure 3C). This finding indicates that Tor was activated under nutrient-rich conditions regardless of the presence of the Big1 protein. That is, induction of autophagy following the addition of IAA did not occur due to inactivation of Tor. To further support the conclusion that Tor is activated under nutrient-rich conditions, we treated the cells with rapamycin for 30 min after 12-h DMSO or IAA treatment. In the control group, rapamycin resulted in a higher mobility of Atg13, Gln3 and Npr1, indicating their dephosphorylation. In the cells with IAA treatment, the bands corresponding to Gln3 and Npr1 shifted to the same position as the control group, suggesting that these two proteins were dephosphorylated to the same level (Figure S4A). However, with rapamycin treatment, even though Atg13 had a higher mobility, it still migrated at a position of higher mass in the BIG1 knockdown cells compared to the control cells, indicating that Atg13 was phosphorylated by kinases other than Tor when Big1 was removed (Figure S4A). In addition, we also noticed a partial phosphorylation of Atg13 in Big1-AID* cells treated with IAA after 30 min of starvation, relative to the control (Figure 3C). To determine if Tor was fully inactive after starvation, we further treated the cells with rapamycin for 20 min following the 30-min starvation. In the cells with and without IAA treatment, rapamycin did not cause further dephosphorylation of Atg13, Gln3 and Npr1 (Figure S4B-D), indicating Tor1 was fully deactivated by 30 min of nitrogen starvation, and Tor was therefore not responsible for the partial phosphorylation of Atg13 under starvation conditions when Big1 was depleted. A similar result was seen with Sch9, another target of Tor [38]. Due to the large size and multiple phosphorylation sites of this protein, we used SDS-PAGE with Phos-tag to better determine the phosphorylation status of Sch9 (Figure S4E and F). Sch9 displayed hyperphosphorylation in the cells without Big1, and rapamycin treatment or nitrogen starvation only resulted in partial dephosphorylation, indicating that Sch9 may be phosphorylated by kinases other that Tor under this condition. All these results indicate that Tor activation only depends on the nutrient status, but not the existence of Big1. A reduced BIG1 expression level can induce autophagy even when Tor was active, and kinases other than Tor appear to be involved in controlling this pathway. Because a previous study suggests that Big1 is important for the integrity of the cell wall [22] we wanted to eliminate the possibility that autophagy was induced due to osmotic stress. To relieve the possible osmotic pressure brought about by Big1 removal, 0.6 M sorbitol was added to the medium to maintain iso-osmotic conditions. However, even with sorbitol present, autophagy was still activated in the Big1-AID* strain after adding IAA (Figure S3C). Therefore, the autophagy induction observed after the knockdown of BIG1 is not likely to result from osmotic stress. Instead, the deficient V-ATPase after Big1 removal may function as a signal to activate autophagy independent from the Tor pathway. A cancer-associated mutation in Big1 induces autophagy From the sequencing data of pure FL B cell DNA isolated from flow sorted FL B cells in FL cases as described in a previous study [16], several mutations in ATP6AP1 were discovered. We focused on two missense mutations that lead to an amino acid change at the N terminus of ATP6AP1 (c.269T>C/T, p.90L>L/P and c.938A>G, p.313Y>C). Sequence alignment showed that the ATP6AP1 L90P and Y313C correspond to yeast Big1 Y75P and F282C, respectively. To study the effect of these mutations on autophagy, we generated these mutations at the genomic BIG1 locus and examined autophagy activity using the GFP-Atg8 processing assay. Cells carrying the Y75P mutation in Big1 showed a higher autophagy flux after starvation for 30 min and 60 min while cells with F282C mutation does not show a significant change compared to the wild type, as quantified by the free to total GFP (GFP + GFP-Atg8) ratio (Figure 3D and E). In addition, we noticed that mutant cells have a lower vacuolar level of some V-ATPase components (Figure S5A), which is consistent with what we observed when BIG1 expression was knocked down using the AID* system. We also tested the V-ATPase in the cells carrying the Y75P mutation in Big1, but did not see significantly reduced activity in these mutant cells even though V-ATPase protein levels on the vacuole decreased (Figure S5B). A possible explanation is that the V-ATPase protein level on the vacuole in the Big1 Y75P mutant was not decreased as severely as seen with Big1 depletion when using the AID system (Fig. 2B). In addition, the Big1 Y75P mutation is chronic compared to the acute depletion of Big1-AID*; thus, the Big1 Y75P mutant cells may have acquired some type of compensation/suppression, and, therefore, the VATPase phenotype would be more difficult to observe. Because we noticed that the Y75P mutation resulted in a lower protein level compared with the wild-type protein (Figure 3D), we decided to determine if the reduced protein level was the only cause of the autophagy phenotype that we observed. To rescue the mutant protein level, we transformed a plasmid carrying CUP1 promoter-driven Big1 Y75P into the cells carrying this mutation at the endogenous BIG1 locus (Figure S5C). The presence of the Big1 Y75P plasmid resulted in a higher protein level of mutant Big1 than seen with the wild-type protein under growing conditions and a similar but slightly higher level during starvation (Figure S5D, left). Even though more mutant protein was present, the autophagy activity was still higher than that seen in the wild-type cells, although it was lower than that of the cells only carrying one copy of the mutant gene (Figure S5D, right). Therefore, we conclude that the Y75P mutation may lead to the instability of this protein and its degradation, which results in a lower protein level, but at the same time this mutation causes a partial loss of function of Big1, together resulting in the increased autophagy phenotype. Overall, we determined that the cancer-associated Y75P mutation in Big1, corresponding to L90P in human ATP6AP1, induces autophagy and disrupts the normal function of the V-ATPase. These results further support the role of Big1 in V-ATPase function and suggest that some cancer-associated mutations in V-ATPase subunits or assembly factors may affect autophagy. ATP6AP1 knockdown induces autophagy in mammalian cells The role of ATP6AP1 in autophagy regulation was also investigated in the human fibrosarcoma cell line HT1080. Here, we used two siRNAs to knock down the expression of ATP6AP1 and found that both led to a significant decrease compared with the cells treated with control siRNA (Figure 4A and B). In both cases, with ATP6AP1 knockdown cells we detected a trend of increasing LC3B-II/Atg8–PE prior to starvation. In addition, compared with the control group, LC3B-II showed a significant increase after starvation (Figure 4A and B). Furthermore, the level of SQSTM1/p62, a receptor that is degraded through the autophagy pathway, was decreased in the ATP6AP1 knockdown cells using siATP6AP1-2 siRNA compared with the control group (Figure 4B). In the case of ATP6AP1 knockdown cells, even though the SQSTM1 protein level was not statistically different from the control group, we still observed a trend of decreasing protein after starvation (Figure 4A). These results demonstrate that knockdown ATP6AP1 induced autophagy in mammalian cells, which is consistent with the observation in yeast cells. Because of the high mutation frequency of ATP6AP1 in multiple human diseases that are closely correlated with autophagy, we explored the role of this V-ATPase assembly factor in autophagy regulation. Through identifying the homolog of ATP6AP1, we focused on Big1, whose function was relatively uncharacterized. In this report, we demonstrated that Big1 plays a critical role in keeping the normal amount and function of V-ATPase on the yeast vacuole and the removal of this protein induces autophagy even under nutrient-rich conditions. Similarly, the knockdown of ATP6AP1 induced autophagy in the HT1080 cell line. We proposed that Big1 may function in controlling V-ATPase activity through interacting with the structural components and assembly factors and we did detect such interactions, including with Vph1 and Vma21 (Figure 2D, S2C and S2D). One feature that distinguishes Big1 with the other known assembly factors is that a large proportion of Big1 is located in the lumen (Figure S6A). To better understand if this lumenal part contributes to Big1 function, we predicted the interaction between Big1 and Vph1 using ColabFold (Figure S6B). In the predicted model with the highest ranking, one interface was identified and this interface was located in the lumenal part of both proteins (Figure S6C). Therefore, we think that Big1 may cooperate with the other assembly factors, interacting with different components in the V0 subunit, thus making the V-ATPase more stable [28, 39]. A more detailed mechanism of how Big1 functions in VATPase assembly or activity may be determined through resolving the structure of this protein. Besides discovering a new role of Big1 in V-ATPase assembly, we think that one of the most interesting discoveries in this report is the simultaneous activation of Tor and autophagy when BIG1 expression is knocked down because Tor is considered as the master negative regulator of autophagy. One explanation of this phenotype is that the removal of the Big1 protein causes deficient V-ATPase, which functions as a signal to induce autophagy. This phenotype is consistent with previous reports; mutations in some V-ATPase components and assembly factors, which result in deficient V-ATPase, lead to increased autophagy activity [16, 17]. However, the downstream effector protein/pathway is unknown and requires further investigation. Besides the hyperphosphorylation of Atg13 and Sch9 during nutrient starvation or after rapamycin treatment (Figure 3C, S4A, S4B, S4E and S4F), we also noticed a mass shift of Atg1 in the yeast cells with BIG1 depleted after starvation, which might indicate an alteration in Atg1 phosphorylation (Figure S4G). Therefore, we speculate that a kinase may be activated when Big1 is removed. Snf1, the AMP-activated serine/threonine protein kinase/AMPK in yeast is involved in autophagy [40]. Accordingly, we tested the activation of Snf1 when Big1 was removed by IAA treatment (Figure S7). Even though we noticed a decreased total protein level in the IAA treatment group, possibly caused by the 3HA tagging [41], the proportion of activated Snf1 (pSnf1) was significantly higher than in the control group (Figure S7B), indicating it could be the cause of hyperphosphorylated Atg13 and Sch9. Thus, hyperactivation of Snf1 may be a reason for the induced autophagy but it may not be the only reason. Therefore, determining the mediators of this change in phosphorylation can shed more light on the autophagy regulation pathway under these conditions. In this study, the regulation of autophagy through ATP6AP1 has been demonstrated and one of the cancer-associated mutations in ATP6AP1 was shown to induce autophagy, suggesting that further analysis can focus on disease-associated mutations in ATP6AP1 [19, 20, 42] to determine whether they cause functional changes in this protein and if the cells carrying these mutations have an abnormal autophagy phenotype. Due to the high mutation frequency of this gene in a wide range of human diseases, these studies will provide us with a deeper understanding of the association between autophagy and human health and may provide new insights in designing new therapeutic strategies.
Acknowledgments
This work was supported by NIH Grant GM131919 to D.J.K.
Author contributions
Y.L., Y.Y., Z.Z., R.Z. and X.S. designed and performed the experiments and interpreted the data. S.N.M., D.T. and D.J.K. designed the experiments. Y.L. and D.J.K. wrote and S.N.M. edited the manuscript.
Declaration of interests
S.N.M. owns shares in Abbvie. The other authors declare no competing interests.
Materials and Methods
Yeast strains, media, and growth conditions Yeast strains used in this study are listed in Table S1. Gene deletions were generated according to the standard method [43]. Strain YYY087 (BIG1[F282C]), YYY088 (BIG1[Y75P]) and ZZH337 (big1 VMA21[QQ]) was made by mutating BIG1 or VMA21 at the corresponding residues on the genome [44]. Yeast cells were cultured at 30°C in rich medium (YPD; 1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal medium (SMD; 0.67% yeast nitrogen base, 2% glucose, and auxotrophic amino acids and vitamins as needed) as indicated. To induce autophagy, cells in the mid-log phase (OD600 = 0.8-1.0) were shifted to nitrogen-starvation medium with glucose (SD-N; 0.17% yeast nitrogen base without ammonium sulfate or amino acids, and 2% glucose) for the indicated times.
Plasmids
To generate the plasmids for the BiFC assay, the CUP1 promoter was amplified from the pCu(416) plasmid [45] and cloned into pRS405 and pRS406 vector plasmids. The VN fragment was PCR-amplified from the pFA6a-VN-TRP1 plasmid [46] and inserted with the PCRamplified ORF of Big1 or Vma21 from the yeast genome after the CUP1 promoter on the pRS405 vector. The VC fragment was PCR-amplified from pFA6a-VC-TRP1 [46] and inserted with the PCR-amplified ORF of VPH1, VMA21, or VOA1 from the yeast genome or alone after the CUP1 promoter on the pRS406 vector. The pRS405-CUP1p-GFP-ATG8 plasmid was constructed by replacing the endogenous ATG8 promoter with the CUP1 promoter and contains the open reading frame of GFP-Atg8 [47]. The pRS406-BIG1p-Big1-PA and pRS414-BIG1pBig1-PA plasmid was constructed by inserting the endogenous BIG1 promoter along with the ORF of BIG1 into the pRS406 and pRS414 plasmid, respectively. The plasmid pRS406-CUP1pBig1-PA was constructed by inserting the CUP1 promoter along with the ORF of BIG1 into the pRS406 plasmid. Similarly, the pRS406-ZEO1p-ATP6AP1 plasmid was constructed by inserting the ZEO1 promoter along with the ORF of ATP6AP1 into the pRS406 plasmid. Before transformation, pRS405-based plasmids were cut with AflII at 37C, and pRS406-based plasmids were cut with BstBI at 65C or StuI at 37C.
AID* system
To set up the , WLY176 (WT) cells were first transformed with the plasmid pNHK53 (ADH1p-OsTIR1-9MYC). Big1 was then tagged with AID-9MYC by homologous recombination. The DNA fragments used for transformation were amplified with pHIS3-AID*9MYC (Addgene, 99524; deposited by Dr. Helle Ulrich) as the template DNA. “AID*” refers to the 71–116 amino acids of the AT1G04250/IAA17 protein in plants [48]. To conditionally knock down BIG1, the cells were first cultured in YPD overnight until mid-log phase and then diluted and treated with 300 μM IAA (Sigma, I2886) for the indicated time to induce the degradation of Big1. For starvation, cells were shifted to SD-N medium, and 300 μM IAA was added to the medium to maintain degradation of Big1. After an appropriate time period of treatment or starvation, samples were collected for western blot, RT-qPCR analysis or vacuole isolation.
Fluorescence microscopy
Cells were cultured in YPD until the mid-log phase (OD600 = 0.8-1.0) and were then harvested and imaged. To label the vacuole as shown in Figure S2B, SynaptoRedC2/FM 4-64 (Biotium) was added at a final concentration of 30 µM. Cells were stained at 30C for 30 min, with shaking every 10 min. After staining, the cells were washed with YPD 3 times and then transferred into YPD for 1 h before imaging. Images were collected on a Leica DMi8 microscope with a 100× objective and Leica Thunder imager. The projection of z-sections is shown in the figure. Vacuole isolation and V-ATPase protein level detection Vacuole isolation followed a previously published protocol [49]. Briefly, after culturing in YPD overnight or after 12-h IAA or DMSO (control) treatment in YPD, approximately 1000 OD600 units of cells were collected and cells were resuspended in 50 ml wash buffer (0.1 M TrisHCl, pH 9.4, 10 mM DTT). The cells were incubated at 30°C in a water bath for 10 min with occasional swirling. The cells were washed once with water and were then suspended in 30 ml spheroplasting buffer (50 mM potassium phosphate [a mixture of 0.8 M K2HPO4 and 0.2 M KH2PO4, pH 7.5] and 1.8 M sorbitol in 0.2 YPD) with gentle vortexing. An appropriate amount of zymolyase (United States Biological, Z1005) was added and the tube was swirled to mix and incubated at 30°C until the OD600 reached 10%-15% of that before the initiation of the zymolyase treatment. The tube was then centrifuged for 2 min at 1000g then the supernatant was removed. Spheroplasts were resuspended in 2.5 mL 15% ficoll (GE Healthcare, 17-0300-10) dissolved in PS buffer (20 mM PIPES, pH 6.8, 200 mM sorbitol). Dextran (200 μL, 2.5 mg/mL; MP Biomedicals, 195133) was added and the tube was put on ice for 3 min then in a 30°C water bath for 5 min. The vacuoles were floated on a four-tiered gradient of ficoll. The gradient was formed by pipetting 2.5-3 ml of the lysate in a 15% ficoll solution in the bottom of the highspeed centrifuge tube (Beckman, 344059). This initial layer was then overlayed with 3 ml 8% ficoll followed by 3 ml 4% ficoll and finally the tube was filled with 0% ficoll (PS buffer). The tubes were centrifuged at 32,000 rpm (Beckman coulter OPTIMA L-90K ultracentrifuge, SW41 rotor) for 90 min at 4°C. The vacuoles were recovered from the interface between the 0% and 4% layers. The total protein amount of the isolated vacuoles was measured using the Pierce Bradford Protein Assay Kit (Thermo Scientific, 23200). After culturing in YPD overnight or after 12-h IAA or DMSO (control) treatment in YPD, 3 OD600 unit cells were collected for total cell lysis preparation. Beads and lysis buffer (20 mM PIPES, pH 6.8, 50 mM KCl, 100 mM KOAc, 10 mM MgSO4, 10 M ZnSO4, 0.5% Triton X-100 [Sigma, T8787]) were added and then the cells were vortexed for 5 min. The protein level in the supernatant was measured using the Pierce Bradford Protein Assay Kit. All of the protein samples (total cell lysis and vacuoles) were diluted in 4% ficoll to the same concentration in a final volume of 100 μl; 20 μl 2MURB (100 mM sodium phosphate pH 7.0, 50 mM MES, pH 7.0, 2% SDS [w:v], 6 M urea, 2 mM NaN3, 2% β-mercaptoethanol, 0.02% bromophenol blue) was then added to each sample. The samples were boiled at 55°C for 15 min. The same amount of samples were loaded and resolved by SDS-PAGE. V-ATPase activity Total ATPase activity was measured using the ATPase Assay Kit (Abcam, ab270551). Bafilomycin A1 (100 nM; Sigma, B1793) was used to specifically inhibit the V-ATPase activity. The V-ATPase activity is considered as the difference before and after BafA1 inhibition. The assay plate was read using a Tecan Microplate Reader Infinite F200 at the wavelength of 660 nm. RNA preparation and RT-qPCR After 12-h IAA or DMSO (control) treatment in YPD and after 1-h starvation, 1 OD600 units of cells were collected. The total RNA for each sample was isolated using the NucleoSpin RNA kit (Macherey-Nagel, 740955). One microgram of total RNA was subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368814). To analyze the cDNA levels, real-time PCR was performed with the Radiant Green Lo-ROX qPCR Kit (Alkali Scientific, QS1005) and run on the QuantStudio5 qPCR Machine (Thermo Fisher). The gene-specific primers for ATG1, ATG7, ATG8, ATG9, and ATG41 and the reference gene TAF10 are listed in previous studies [30, 50].
Colony dot blot
The cells were cultured in YPD until mid-log phase (OD600 = 0.8-1.0) and yeast cells were collected and diluted to 1.0 OD600/ml; eight-fold serial dilutions were made in water. A 1.5 μl aliquot of each dilution was spotted on a YPD plate at pH 5 or pH 7.5 containing 35 mM CaCl2. Co-immunoprecipitation Cells were grown in 60 ml SMD -Trp medium at 30°C overnight. Cells (60 OD600 units) were harvested and washed with ice-cold 1 PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). The cell pellet for each sample was resuspended in 150 uL IP buffer (1 PBS, 200 mM sorbitol, 1 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF and 1 tablet of proteinase inhibitor [Roche] in 50 mL IP buffer) and separated into four tubes. Glass beads were added into each tube and the samples were mixed by vortexing at 4°C and then centrifuged at 16,110 g for 10 min. A 60-µL aliquot was taken from each sample as the input, and 1.2 mL supernatant was incubated with IgG Sepharose beads (GE Healthcare Life Sciences) at 4°C for 2 h. After incubation, the beads were washed with IP buffer 6 times. Next, 50 µL 2 MURB (100 mM sodium phosphate, pH 7.0, 50 mM MES, pH 7.0, 2% SDS [w:v], 6 M urea, 2 mM NaN3, 2% β-mercaptoethanol, 0.02% bromophenol blue) was added to the beads and the beads were heated at 55°C for 15 min. The supernatants were resolved by SDS-PAGE. For the input samples, 1 mL 10% TCA was added, and the tube was placed on ice for 30 min. The input samples were then centrifuged at 16,110 g and the pellets were washed with acetone and air- dried. Fifty µL 1 MURB and glass beads were added to the pellet, followed by vortexing for 5 min. The samples were then heated at 55°C for 15 min and resolved by SDS-PAGE. Western blot, antibodies and antisera Western blot was performed as described previously [51]. In Figure S7A, p-Snf1was first blotted and then the membrane was washed twice with stripping buffer (1.5% glycine, 0.1% SDS, 1% Tween-20, pH 2.2) for 10 min at room temperature. Next, the membrane was washed with 1PBS twice for 10 min each time, followed by two washes for 5 min each with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20). After blocking with 5% milk in TBST, anti-HA antibody was used to blot total Snf1. Anti-Pgk1 antiserum is a generous gift from Dr. Jeremy Thorner (University of California, Berkeley). Other antibodies were as follows: YFP, which detects GFP (Clontech, 63281), MYC/c-Myc (Sigma, M4439), Vph1 (Abcam, ab113683), Vma2 (Abnova, MAB14189), Prc1/CPY (Invitrogen, A-6428), HA (Sigma-Aldrich, H3663), PA (ImmunoResearch, 323-005- 024), Dpm1 (Invitrogen, A-6429), p-Snf1 (Cell Signaling Technology, 2531), goat anti-rabbit IgG secondary antibody (Fisher, ICN55676) and rabbit anti-mouse IgG secondary antibody (Jackson, 315-035-003). Anti-Vma8 [52], anti-Vma4 [52] and anti-Atg1 [53] antibody were described previously.
Mammalian cell methods
HT1080 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, 11995073) supplemented with 10% heat-inactivated fetal bovine serum (Millipore, TMS-013-B) and 1% penicillin and streptomycin (Thermo Fisher Scientific, 15070-063) at 37°C, 95% humidity, and 5% CO2. These cells were transfected with ATP6AP1-specific siRNAs (Sigma, SASI-Hs01-0015-1228 and SASI-Hs01-0015-1229) or a universal negative control (Sigma, SIC001) using Lipofectamine RNAiMAX (Invitrogen, 13778075). Transfections involved 100 pmol of siRNA per well in 6-well plates. After 48 h, protein levels were assessed by western blot using antibodies against ATP6AP1 and ACTB. To induce starvation, we used Hanks’ balanced salt solution (HBSS; Thermo Fisher Scientific, 24020117) in the absence or presence of bafilomycin A1 (Selleck Chemicals, S1413) after transfection for 48 h. Samples were collected after 0, 2, and 4 h of starvation and the protein levels were evaluated by western blot. Antibodies used were as follows: ATP6AP1 (Thermo fisher, PA5-37328; 1:1000), ACTB (Cell Signaling Technology, 3700; 1:5000), SQSTM1 (Sigma, p0067; 1:1000), MAP1LC3/LC3 (Cell Signaling Technology, 4108; 1:1000). Protein structure and interaction prediction The predicted structure of Big1 was obtained from the AlphaFold database (https://alphafold.ebi.ac.uk/). The interaction between Big1 and Vph1 was predicted using ColabFold [54]. The model with the highest ranking is shown in the figure.
Statistical analysis
A two-tailed t-test was used to determine statistical significance.
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