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The entry of unclosed autophagosomes into vacuoles and its physiological relevance [1]

['Zulin Wu', 'College Of Life Sciences', 'Key Laboratory Of Agricultural Environmental Microbiology Of Ministry Of Agriculture', 'Rural Affairs', 'Nanjing Agricultural University', 'Nanjing', 'Haiqian Xu', 'Pei Wang', 'National Laboratory Of Biomacromolecules', 'Cas Center For Excellence In Biomacromolecules']

Date: 2022-12 

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

Normal autophagy is critical for cellular homeostasis, whereas defective autophagy is closely linked to human disease development and progression. The model organism yeast has been greatly contributing to basic and applied researches of autophagy. Previously, we found that unclosed double-membrane APs accumulated as clusters outside vacuoles in haploid yeast Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. If these APs could not enter vacuoles to be degraded, the autophagy in these mutants would be completely interrupted to be defective. However, we found herein that the unclosed double-membrane APs in Vps21-mutant cells entered vacuoles to be degraded after prolonged autophagy induction or to be unclosed single-membrane autophagic bodies if further lacking vacuolar hydrolases. When we cultured diploid Vps21-mutant cells in a sporulation medium, their unclosed APs also entered vacuoles to be degraded, so that these diploid mutant cells could progress through meiosis to sporulation as diploid wild-type cells, not to be complete infertile as diploid cells of typical autophagy mutants. VTC proteins, Ypt7, and Vam3 were all important for the entry of unclosed double-membrane APs into vacuoles. Facilitating the entry of unclosed APs into lysosomes for degradation through autophagy may serve as a remedy strategy in autophagosome-closure-impaired diseases.

Funding: This work was supported by grants from the Natural Science Foundation of China grant numbers 91954125, 31871428, 31671479 to Y. L. ( https://isisn.nsfc.gov.cn ), the National Key R&D Program of China grant number 2017YFA0504700 to Y. X. ( https://service.most.gov.cn/ ), and the open funding from the State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University grant number MMLKF21-06 to Y. L. & Z. X. ( https://skmml.sjtu.edu.cn/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we induced the accumulation of unclosed APCs outside vacuoles in Vps21- or ESCRT-mutant cells, with or without vacuolar proteases, and then assessed the fate of the unclosed APCs after a prolonged period of nitrogen starvation or rapamycin treatment. Using cellular and biochemical methods, we demonstrated that unclosed double-membrane APCs can ultimately enter vacuoles and become unclosed single-membrane ABs after prolonged nitrogen starvation. Furthermore, we found that the vacuolar proteases Pep4 and Prb1, several VTC proteins, the Rab GTPase Ypt7, and the SNARE Vam3 played important roles in affecting the entry of unclosed double-membrane APCs into vacuoles. This process contributed to meiosis and sporulation in diploid Vps21-mutant cells. Thus, we discovered an unexpected phenomenon that unclosed double-membrane APCs in yeast could enter vacuoles after a prolonged period of autophagy induction. This process prevents autophagy from being completely interruption and restores the cells to some level of normal physiology, such that sporulating diploid Vps21-mutant cells can survive under induced stress.

In our previous studies [ 10 , 11 ], we observed a few interesting phenotypes in Vps21- or ESCRT-mutant cells after nitrogen starvation: 1) not all mutant cells in the same culture displayed APC accumulation, 2) older cultures before nitrogen starvation were less likely to accumulate APCs, 3) a higher percentage of mutant cells accumulated APCs when they lacked the vacuolar hydrolase Pep4, and 4) APCs were occasionally seen inside vacuoles. As the expression levels of yeast vacuolar proteases change in a growth-stage-dependent manner and peak when yeast cells approach the stationary phase [ 12 , 13 ], it is possible that the older cultures of Vps21- or ESCRT-mutant cells expressed higher levels of vacuolar proteases. Increased levels of vacuolar proteases might promote the entry of APCs into vacuoles. Conversely, the absence of vacuolar proteases in Vps21- or ESCRT-mutant cells might inhibit the entry of APCs into vacuoles. Our observations suggest that the entry of unclosed APCs into vacuoles might depend on the levels of vacuolar proteases, contrary to the paradigm that only closed, mature APs can enter lysosomes/vacuoles through fusion.

Previously, we found that AP closure was impaired in yeast Vps21- or ESCRT (the endosomal sorting complex required for transport)-mutant cells, in which unclosed APs accumulated as clusters around vacuole membranes (mostly outside the vacuoles) shortly after autophagy was induced (i.e., nitrogen starvation for 2 h or treatment with rapamycin for 4 h) [ 10 , 11 ]. However, it remains to be determined whether the unclosed autophagosome clusters (APCs) can enter vacuoles for degradation.

Autophagy involves multiple steps, including initiation, phagophore expansion, closure, fusion, and cargo degradation in lysosomes/vacuoles [ 1 , 2 ]. Impaired or defective autophagy is closely linked to disease development and progression [ 3 ]. It has been widely described in the literature that the outer membrane of closed mature double-membrane autophagosomes (APs) fuses with the lysosome/vacuole membrane. Then, the closed AP only containing the inner membrane is thought to be released into the lysosome/vacuole to form a single-membrane autophagic body (AB), which is further degraded by hydrolases and recycled [ 4 , 5 ]. However, the above-described fusion step has not been clearly or thoroughly demonstrated through experimentation. Previous transmission electron microscope (TEM) data showed that, in yeast, the double-membrane AP structures contacted the vacuole membrane and occasionally formed a continuous membrane between the AP membrane and the vacuole membrane [ 6 ]. However, the double membrane of APs and single membrane of ABs were not clearly shown in that study due to the resolution limit of conventional TEM. Thus, it was unclear whether only the outer membrane of APs (but not the inner membrane or both membranes) dynamically fused with lysosome/vacuole membranes in live cells. Fusion of the outer membrane of closed, mature double-membrane APs with lysosome/vacuole membranes requires additional experimental support, given that the double membrane of APs and single membrane of ABs can be observed clearly and dynamically. In addition, only closed APs were proposed to fuse with vacuoles/lysosomes as closed APs accumulated in yeast ypt7Δ or vam3Δ cells or the corresponding mammalian AP-lysosome fusion mutant lines [ 7 – 9 ], although no experimental evidence excludes the possibility that unclosed APs can enter vacuoles. Therefore, it remains unknown whether unclosed APs (i.e., phagophores) can fuse with lysosome/vacuole membranes to deliver unclosed APs into lysosomes/vacuoles for degradation.

Results

Growth-stage- and hydrolase-dependent accumulation of unclosed APCs in vps21Δ cells after a short period of autophagy induction In the first two remarkable yeast autophagy-mutant screenings, relatively few autophagy mutants were screened out [14,15]. Clearly, some autophagy-related proteins were missed from these two screenings, including small GTPases and other proteins [16,17]. Previously, we found that the absence of the Rab5 GTPase Vps21 resulted in the accumulation of phagophore clusters (i.e., unclosed APCs) mostly outside vacuoles after 2 h of nitrogen starvation in SD-N medium [11,18]. However, when cultures with high optical density at 600 nm (OD 600 ) values were subjected to nitrogen starvation, the percentages of Vps21-mutant cells with accumulated APCs decreased [11]. These observations suggest that APC accumulation after nitrogen starvation was related to the growth stage. As an autophagosome marker in yeast, the green fluorescent protein (GFP)-Atg8 is delivered to vacuoles through autophagy and degraded to the stable GFP in wild-type (WT) cells. The resulting localization of GFP-Atg8 can be observed by fluorescence microscopy. The yeast precursor form of aminopeptidase I (prApe1) is proteolytically cleaved upon vacuolar delivery or during non-selective autophagy to the mature form of Ape1 (mApe1) in WT cells. The resulting shifts in molecular mass from GFP-Atg8 to GFP or from prApe1 to mApe1 can be monitored by immunoblotting assays [19,20]. To clearly demonstrate the effect of growth-stage on APC accumulation in vps21Δ cells, we diluted cells cultured overnight in yeast extract peptone dextrose (YPD) medium to an OD 600 value of 0.05, after which they were grown to OD 600 values of 0.5, 1, 2, 3, or 4. The cells were stained with FM4-64 dye for 1 h before they were collected for fluorescence microscopy observations. Comparable localizations of GFP-Atg8 to the cytosol but not to FM4-64-labelled vacuoles were observed in WT cell (OD 600 = 1) and vps21Δ cells (at different OD 600 values) (Fig 1A). Immunoblotting analysis showed that GFP-Atg8 was not degraded in WT or vps21Δ cells under any conditions investigated (Fig 1B). In contrast, prApe1 did not mature to mApe1 in vps21Δ cells due to defective vacuolar delivery under normal growth conditions, whereas prApe1 partially matured in WT cells (OD 600 = 1) (Fig 1B). Because GFP-Atg8 entry into vacuoles in WT cells was not affected by varying the cell density (OD 600 = 0.5–4, before nitrogen starvation) (Fig 1A and 1B), we only presented data for WT cells grown to OD 600 = 1 from here onward. Interestingly, when vps21Δ cells grown to various cell densities (OD 600 = 0.5–4) were adjusted to a similar cell density (OD 600 = ~0.8–1) and subjected to nitrogen starvation for 2 h, the cells starting from the lowest cell density (OD 600 = 0.5) showed the highest percentage (94%) of cells with APCs around the vacuole membranes, whereas those starting from the highest cell density (OD 600 = 4) showed the lowest percentage (~20%) (Fig 1C). Moreover, the number of cells with GFP-positive vacuoles increased as the cell density increased (Fig 1C). GFP-Atg8 degradation and prApe1 maturation in vps21Δ cells increased as the cell density increased, suggesting that autophagy became elevated (Fig 1D). These data indicate that, although the late growth stage itself before nitrogen starvation did not result in increased autophagy, vps21Δ cells at a late growth stage are able to accumulate fewer APCs after a short period of nitrogen starvation. PPT PowerPoint slide

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TIFF original image Download: Fig 1. The growth stage before nitrogen starvation and the availability of vacuolar hydrolases impacted the accumulation of autophagosome clusters (APCs) in vps21Δ cells. A. The increased cell density (OD 600 ) of vps21Δ cells grown in rich medium did not result in GFP-Atg8 entering into vacuoles. GFP-Atg8-labeled WT and vps21Δ cells were grown to the indicated OD 600 values in rich medium (yeast extract peptone dextrose [YPD] medium) and observed for GFP-Atg8 localization and FM4-64 signals. For WT cells, only the results at an OD 600 of 1 are presented. B. No autophagy processing was detected in vps21Δ cells with OD 600 values ranging from 0.5 to 4. The cells were grown as described in panel A. GFP-Atg8 processing to GFP and prApe1 processing to mApe1 were determined for cell lysates by performing immunoblotting analysis with anti-GFP and anti-Ape1 antibodies, respectively. G6PDH was detected as a loading control. C. When starting with a high cell density before nitrogen starvation, the accumulation of GFP-Atg8-labeled APCs in vps21Δ cells decreased after nitrogen starvation. WT and vps21Δ cells were grown and starved as indicated and stained with FM4-64 for 1 h before being harvested for fluorescence observations. The percentages of cells containing APCs were quantified and are presented below the merged pictures. D. When the cell density was high before nitrogen starvation, autophagy processing in vps21Δ cells increased after nitrogen starvation. Cells were grown as described in panel C. GFP-Atg8 and prApe1 processing were determined for cell lysates as described in panel B. G6PDH was detected as a loading control. GFP-Atg8 and prApe1 processing were quantified and are presented below the G6PDH blot. E. The accumulation of GFP-Atg8-labeled APCs in vps21Δ cells increased after nitrogen starvation in the absence of the vacuolar hydrolases Pep4 and Prb1. Cells were grown as described in panel A to an OD 600 of 2 and starved in SD-N medium for 2 h. The cells were examined and the data are presented as described in panel C. F. The partial autophagy processing observed in vps21Δ cells after nitrogen starvation was completely blocked in vps21Δpep4Δprb1Δ cells. The cells were grown as described in panel E, and autophagy processing was determined and presented as done in panel D. PhC, phase contrast; scale bars in panels A, C, and E, 5 μm; arrows, APCs, and OD, OD 600 . The data shown are presented as the mean +/- the standard deviation (STD). **p < 0.01; ***p < 0.001. Over 600 cells per strain were counted. The results shown represent three independent experiments. https://doi.org/10.1371/journal.pgen.1010431.g001 Previous reports showed that the levels of vacuolar proteases changed in a growth-stage-dependent manner and peaked when the cells approached the stationary phase [12,13]. Therefore, the decreased APC accumulation in vps21Δ cells starting from the high cell density might have been partially caused by increased vacuolar protease levels. Conversely, a decreased level or absence of vacuolar proteases might have led to increased APC accumulation. Indeed, we noticed that the percentages of Vps21- or ESCRT-mutant cells displaying accumulated APCs was higher in the absence of the Pep4 hydrolase [10,11,18]. To confirm these observations, we constructed WT and vps21Δ strains that lacked two key vacuolar proteases (Pep4 and Prb1). Then, we examined the status of GFP-Atg8 localization and autophagy processes after the cells were intentionally cultured to a relatively high cell density (OD 600 = 2) and subjected to nitrogen starvation for 2 h. The percentage of cells displaying APCs increased from 66% in vps21Δ cells to 95% in vps21Δpep4Δprb1Δ cells under the same growth conditions (Fig 1E). As expected, autophagy was completely blocked in vps21Δpep4Δprb1Δ cells, similar to the results found with pep4Δprb1Δ and atg1Δ cells (Fig 1F). These results confirmed the roles of vacuolar hydrolases in APC accumulation in vps21Δ cells after a short period of nitrogen starvation. Collectively, these findings indicate that unclosed APC accumulation in vps21Δ cells depended on the growth stage and expression levels of hydrolases. A high percentage of vps21Δ cells lacking hydrolases could still accumulate APCs at the late growth stage. These data suggest that some APCs in vps21Δ cells might have entered vacuoles when the cells were during the late growth stage.

The physiological relevance of the entry of unclosed APCs into vacuoles in vps21Δ cells during nitrogen starvation Yeast autophagy is essential for starvation resistance and sporulation. The absence of core autophagy-machinery proteins, such as Atg1, resulted in decreased cell viability under nitrogen starvation in yeast haploid cells and infertility in diploid cells [14,15]. Starvation resistance and sporulation assays often take a few days of monitoring, whereas accumulated APCs in vps21Δ cells started to enter vacuoles between 2 and 8 h (even in vps21Δpep4Δprb1Δ cells) and most accumulated APCs ultimately entered vacuoles. It is impossible to compare transient physiological differences between vps21Δ cells containing APCs outside vacuoles after approximately 2 h of nitrogen starvation and those containing APCs inside vacuoles after approximately 8 h of nitrogen starvation using either of these assay methods. However, if autophagy was completely blocked because the unclosed APCs could not enter vacuoles for degradation in vps21Δ/vps21Δ (diploid) cells, then the cells would not sporulate, just like atg1Δ/atg1Δ and pep4Δ/pep4Δ cells, in which autophagy was not initiated or fulfilled, respectively. Otherwise, if vps21Δ/vps21Δ cells were not infertile as atg1Δ/atg1Δ and pep4Δ/pep4Δ cells were under sporulation conditions, then it would be certain that autophagy in vps21Δ/vps21Δ cells was not completely blocked (i.e., the accumulated APCs in vps21Δ/vps21Δ cells entered vacuoles resulting in proliferation). We examined the sporulation ability of vps21Δ/vps21Δ cells, with WT/WT, pep4Δ/pep4Δ, and atg1Δ/atg1Δ cells used as controls. In vps21Δ/vps21Δ cells, GFP-Atg8 also accumulated strongly in APCs after 2 h of nitrogen starvation (Fig 9A, top), and GFP-Atg8 degradation was completely blocked (Fig 9B). Autophagy was not efficiently induced after growth in sporulation medium (SPO) for 2 h with all tested cell lines. However, 4 or 8 h was sufficient to induce autophagy in all tested cells except for atg1Δ/atg1Δ cells. GFP-Atg8 entered vacuoles in WT/WT cells. Puncta but not APCs were observed in vps21Δ/vps21Δ and vps21Δ/vps21Δ pep4Δ/pep4Δ cells grown in SPO for 4 h. The puncta disappeared in vps21Δ/vps21Δ cells although puncta or APCs were observed in vps21Δ/vps21Δ pep4Δ/pep4Δ cells grown in SPO for 8 h (Fig 9A, middle). When the diploid cells were grown in SPO for 3 days, tetrads were observed in over 60% of WT/WT cells but not in pep4Δ/pep4Δ and atg1Δ/atg1Δ cells. Tetrads were easily observed in approximately 60% of vps21Δ/vps21Δ cells. GFP signals regularly distributed in the spores and ascal cytosol of tetrads. The percentage of WT/WT cells with tetrads increased to approximately 80% after growth in SPO for 14 days, whereas no significance was found regarding the percentage of vps21Δ/vps21Δ cells with tetrads (Fig 9A and 9C). After growth in SPO for 14 days, the GFP signals in tetrads decayed more in WT/WT cells than in vps21Δ/vps21Δ cells, whereas GFP-Atg8 degradation was almost complete in both WT/WT and vps21Δ/vps21Δ cells after growth in SPO for 3 days (Fig 9A and 9B). The sporulation ability was completely abolished in vps21Δ/vps21Δ pep4Δ/pep4Δ cells (Fig 9A and 9C). Although the combination of pep4Δ/pep4Δ should delay the entry of unclosed APCs into vacuoles in vps21Δ/vps21Δ cells, the unclosed APCs ultimately entered vacuoles and were not degraded due to the absence of the vacuolar hydrolase Pep4. GFP-Atg8 degradation in vps21Δ/vps21Δ pep4Δ/pep4Δ cells was similar to that in pep4Δ/pep4Δ cells (Fig 9B). The partial degradation of GFP-Atg8 in sporulation-defective strains (such as the atg1Δ/atg1Δ and pep4Δ/pep4Δ strains) grown in SPO for 3 days did not induce sporulation (Fig 9 and [32]). We propose that Atg1-independent proteolysis occurs that may be proceeded by vacuolar hydrolases when cells are grown in SPO. This possibility should be investigated in the future. Our results indicate that the entry of unclosed APCs into vacuoles in vps21Δ/vps21Δ cells and their complete degradation with vacuolar hydrolases led to sporulation; otherwise, they should have behaved like the infertile atg1Δ/atg1Δ and pep4Δ/pep4Δ cells. PPT PowerPoint slide

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TIFF original image Download: Fig 9. Diploid vps21Δ/vps21Δ cells could sporulate in sporulation medium (SPO). A. The vps21Δ/vps21Δ cells were capable of sporulation like WT/WT cells. The indicated diploid cells were grown in SD-N medium for 2 h (top), SPO for different numbers of h in the first day (middle), or SPO for days (bottom), after which PhC and GFP fluorescence images were obtained. GFP-Atg8-labeled clusters clearly accumulated in vps21Δ/vps21Δ cells in SD-N medium. Scale bars, 5 μm; arrows, APCs. B. Immunoblotting analysis of the autophagy process in the indicated diploid cells grown in SD-N medium for 2 h (left) or SPO for 3 days (right). The cells were grown as described in panel A and collected for immunoblotting analysis as described in Fig 1B. C. The percentages of cells with tetrad spores shown in panel A after sporulation proceeded for 3 and 14 days. n.s., not significant; *p < 0.05; ***p < 0.001. The results shown represent at least two independent experiments. https://doi.org/10.1371/journal.pgen.1010431.g009

Depleting of vacuolar hydrolases exacerbates unclosed APC accumulation around vacuole membranes in ESCRT mutants, and APCs enter vacuoles after prolonged nitrogen starvation Unclosed APCs accumulated around vacuole membranes in most cells of ESCRT- subunit mutants [10]. We wondered whether those APCs might enter vacuoles after prolonged nitrogen starvation. Thus, we observed GFP-Atg8 and FM4-64 fluorescence in representative ESCRT mutants (snf7Δ cells and vps4Δ cells) lacking two key vacuolar hydrolases (Pep4 and Prb1) after 2 h of nitrogen starvation. We confirmed that increased APC accumulation occurred in ESCRT mutants lacking both vacuolar hydrolases (S5A Fig and [10]). As expected, the partial GFP-Atg8 degradation and prApe1 maturation in ESCRT-mutant cells were impaired when PEP4 and PRB1 were further deleted (S5B Fig). We also examined GFP-Atg8 and FM4-64 localizations in snf7Δ and snf7Δpep4Δprb1Δ cells after prolonged nitrogen starvation. We found that the percentage of cells with GFP-Atg8 clusters in snf7Δpep4Δprb1Δ cells was significantly higher than that in snf7Δ cells between 2–6 h of nitrogen starvation. APCs in snf7Δ and snf7Δpep4Δprb1Δ cells gradually entered vacuoles between 2–6 h, and this phenomenon occurred faster in snf7Δ cells (S5C and S5D Fig). The GFP-Atg8-labeled APCs also gradually entered vacuoles in vps4Δ cells after prolonged nitrogen starvation. It is noteworthy that not all APCs entered vacuoles and became completely degraded, as ~40% of GFP-Atg8 and ~40% of prApe1 in vps4Δ cells were not processed even after 24 h of nitrogen starvation (S6 Fig), although more time might have been needed for the degradation to occur. Ultrastructural analysis by TEM showed that AP-related membrane structures appeared inside vacuoles in ESCRT-mutant cells or cells further lacking Pep4 and Prb1 after 8 h of nitrogen starvation, where a significantly higher percentage of cells displayed APCs inside vacuoles than outside vacuoles (S7A and S7B Fig). Protease-protection assays showed that these AP-related membrane structures were unclosed in ESCRT-mutant cells or cells further lacking Pep4 and Prb1 after either 2 or 8 h of nitrogen starvation (S7C and S7D Fig). These results were similar to those in vps21Δ and vps21Δpep4Δprb1Δ cells under the same conditions (Figs 2, 3 and 6). Therefore, we propose that AP-related membrane structures inside vacuoles of ESCRT-mutant cells or cells further lacking Pep4 and Prb1 were unclosed single-membrane ABs converted from unclosed double-membrane APCs outside vacuoles.

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