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1 学位論文の内容の要旨 論文提出者氏名 蒋培都 論文審査担当者 主査清水重臣副査中田隆夫 淺原弘嗣 論文題目 The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. ( 論文内容の要旨 ) Background Macroautophagy (referred to as autophagy hereafter) is an intracellular degradation system that delivers cytoplasmic components such as large molecules and organelles to lysosomes. Autophagy consists of several sequential steps: autophagosome formation, autophagosome lysosome fusion, breakdown of autophagic cargo and efflux, and autophagic lysosome reformation. Dysregulation of autophagy has been implicated in aging, innate immunity, and various human diseases including cancers, neurodegenerative diseases, and immune disorders. To date, the molecular mechanism regulating the fusion of autophagosomes with lysosomes has not yet been fully established. Most of the autophagy-related (Atg) genes indentified so far mainly function in the autophagosome formation step. It is likely that the canonical intracellular fusion machinery composed of Rabs, SNAREs, and tethers controls autophagosome lysosome fusion. It has been shown that the Rab GTPase Rab7 regulates the maturation of late autophagic vacuole dependent on its GTPase activity. Recently, our group reported that the Qa-SNARE syntaxin 17 (STX17) is inserted into the completed autophagosomes via its unusual C-terminal hairpin-like structure and mediates autophagosome lysosome fusion by binding to its partner SNAREs: SNAP29 (cytosolic Qbc-SNARE) and VAMP8 (endo/lysosomal R-SNARE). On the other hand, the involvement of tethering factors acting between the autophagosome and lysosome remains less clear. Homotypic fusion and protein sorting (HOPS), is a conserved protein complex consisting of Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41. It was originally identified in yeast as a tethering complex that is now known to be generally important for vacuolar/lysosomal fusion events from yeast to mammals. Therefore, it is necessary to clarify whether, and if so how, HOPS functions in the autophagy pathway. Methods Cell culture HeLa cells, human embryonic kidney (HEK) 293T cells, and mouse embryonic fibroblasts - 1 -

2 (MEFs) were cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal bovine serum, 50 g/ml penicillin, and streptomycin (regular medium) in a 5% CO 2 incubator. For starvation treatment, cells were washed with phosphate-buffered saline (PBS) and incubated in amino acid-free DMEM without serum (starvation medium). RNA interference Two independent Stealth RNAi oligonucleotides (Invitrogen) were transfected into cells using Lipofectamine RNAi MAX ( ; Invitrogen) according to the manufacturer s instructions. After 2 days, the cells were again transfected with the same sirna and cultured for an additional 3 days before analysis. Immunoprecipitation and immunoblotting For immunoprecipitation analysis, HEK293T cells were transiently transfected with plasmids using FuGENE 6 reagent ( ; Roche Applied Science). Cells from each samples were suspended in lysis buffer containing 0.3% CHAPS. Cell lysates were then analyzed by immunoprecipitation using anti-flag M2 affinity gel (50% slurry; Sigma-Aldrich) or anti-gfp antibody in combination with protein G-Sepharose (GE Healthcare). For immunoblotting analysis, cells were lysed with lysis buffer containing 1% Triton X-100. Immunocytochemistry Cells grown on coverslips were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were permeabilized with 50 μg/ml digitonin in PBS for 5 min, blocked with 3% bovine serum albumin in PBS for 30 min at room temperature, and incubated with primary antibodies for 1 h. After PBS washing, cells were incubated with fluorescence-conjugated secondary antibodies for another 1 h. Images were acquired on a confocal laser microscope (FV1000D IX81; Olympus) using a 60 PlanApoN oil immersion lens (1.42 NA; Olympus), and captured with Fluoview software (Olympus). EGFR degradation assay HeLa cells were plated at 80% confluence, starved in serum-free DMEM overnight, and then incubated with DMEM supplemented with 100 ng/ml EGF ( ; Wako Chemicals). Cells were rinsed with PBS twice and lysed in lysis buffer containing 1% Triton X-100. Electron microscopy HeLa cells were cultured on collagen-coated plastic coverslips (Cell tight C-1 Cell disk; Sumitomo Bakelite) and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (ph 7.4) for 2 h. The cells were washed in the same buffer three times, post-fixed in 1.5% osmium tetroxide in 0.1 M phosphate buffer for 2 h, dehydrated, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and observed using a Hitachi H-7100 electron microscope. Statistical analysis Numerical data are presented as mean ± SEM values. Statistical comparisons were made using the two-tailed Student s t test, unless otherwise indicated

3 Results STX17 interacts with the HOPS complex To search for proteins that function in the autophagosome lysosome fusion step together with STX17, human FLAG-tagged STX17 was expressed in HEK293T cells, immunoprecipitated with anti-flag antibody, and subjected to highly sensitive direct nano-flow liquid chromatography/tandem mass spectrometry (LC MS/MS). As a result, two HOPS components, VPS33A and VPS16, were identified as STX17-binding proteins. We confirmed that FLAG-STX17 interacts with Myc-tagged VPS33A and GFP-tagged VPS16 by immunoprecipitation. Additionally, interactions between STX17 and all other HOPS components VPS11, VPS18, VPS39, and VPS41 were detected. Furthermore, endogenous VPS33A and VPS16 were precipitated with GFP-STX17. HOPS translocates to STX17-positive autophagosomes Under growing conditions, GFP-VPS33A localized mainly to LAMP1-positive endo/lysosomes and did not colocalize with STX17. However, Myc-STX17 formed punctate structures under starvation conditions, and most of them colocalized with the autophagosomal marker LC3. Moreover, GFP-VPS33A translocated to these STX17- and LC3-positive structures. But GFP-VPS33A did not colocalize to ATG16L1-positive isolation membranes (also known as phagophores). VPS16 also colocalized with STX17 and LC3. These data suggest that the HOPS components are recruited to completed autophagosomes where STX17 is also localized. The HOPS complex is required for autophagic activity When the HOPS component VPS33A was silenced with sirnas, LC3-II accumulated, even under growing conditions. The LC3-II level was not further increased by treatment of the vacuolar ATPase inhibitor bafilomycin A1, suggesting impaired LC3-II turnover in these cells. Moreover, starvation-induced degradation of p62, a selective autophagy substrate, was suppressed in these cells, indicating that autophagic flux is blocked. Expression of sirna-resistant VPS33A, which has silent mutations in the sirna target sequence, restored the impaired autophagic flux caused by VPS33A knockdown, confirming that the phenotype of VPS33A depletion is not due to an off-target effect. Furthermore, knockdown of other HOPS components, VPS16 and VPS39, phenocopied the effect of VPS33A depletion. These results suggest that HOPS is required for autophagic activity and probably functions in a late step of the autophagy pathway. The HOPS complex is required for autophagosome lysosome fusion In control sirna-treated cells, a high rate of LC3 LAMP1 colocalization was observed following 24-h serum starvation in the presence of the lysosomal inhibitors. However, the LC3 LAMP1 colocalization rate was notably lower in VPS33A and STX17 sirna-treated cells. Moreover, knockdown of VPS33A or another HOPS component VPS39 caused accumulation of STX17- and LC3-double positive LAMP1-negative structures, which should represent complete autophagosomes, under either growing or starvation conditions, suggesting a defect in both basal and starvation-induced autophagy. Consistent with the immunofluorescence data, electron microscopy showed accumulation of autophagosomes in VPS33A or STX17 knockdown cells. Importantly, most of these autophagosomes were not degraded. Taken - 3 -

4 together, these data suggest that the HOPS complex is a bona fide fusion factor in the autophagy pathway. Endocytic pathway is affected in VPS33A knockdown cells but not in STX17 knockdown cells Degradation of EGFR following EGF stimulation was delayed in VPS33A knockdown cells. However, this delay was not observed in STX17 knockdown cells. EGF was trapped in EEA1-positive endosomes in VPS33A knockdown cells, but was normally delivered to LAMP1-positive endo/lysosomes in STX17 knockdown cells. These data suggest that HOPS is required for both endocytosis and autophagy (autophagosome lysosome fusion), whereas STX17 is specifically required only for the latter. UVRAG is not directly involved in autophagosome lysosome fusion Knockdown of ultraviolet irradiation resistance-associated gene (UVRAG), a known HOPS-interacting protein, did not significantly decrease the LC3 LAMP1 colocalization in the presence of lysosomal inhibitors, suggesting that UVRAG is dispensable for autophagosome lysosome fusion. Furthermore, no STX17-positive structures accumulated in UVRAG knockdown cells, while LC3- and LAMP1-double positive structures did accumulate. Discussion HOPS complex is a bona fide autophagosomal fusion factor In the present study, we demonstrated that the HOPS complex is a bona fide autophagosomal fusion factor, which interacts with the autophagosomal SNARE, STX17. Although STX17 is required for autophagosome lysosome fusion but not for the endocytic degradation, HOPS is required for both processes. Our current model for autophagosome lysosome fusion is as follows. In normal growing cells, the HOPS complex is localized mainly to endo/lysosomes, whereas STX17 is present in ER, mitochondria, and cytosol. Upon autophagy induction, the isolation membranes elongate to form completed autophagosomes. Then, STX17 translocates to these autophagosomes, where HOPS is also recruited. HOPS bridges closely apposed autophagosomal and lysosomal membranes and facilitates the assembly of the trans-snare complex (STX17-SNAP29-VAMP8) to mediate autophagosome lysosome fusion. UVRAG, a known HOPS-interacting protein, is not directly involved in autophagosome lysosome fusion The present results demonstrate that, although UVRAG is indeed required for a late step in endocytic degradation, it is not directly involved in autophagosome formation or autophagosome lysosome fusion. The apparent effect of UVRAG on autophagic degradation may be secondary to its role in endocytic degradation. In fact, any defects in the endocytic pathway could also affect the autophagy pathway. Conclusion HOPS promotes autophagosome lysosome fusion through interaction with STX17 in addition to its well-established function in the endocytic pathway

5 < 和文による要約 > 細胞内では様々な膜輸送が行われており 膜同士の融合は SNARE 分子 Rab 分子 tethering 複合体によって厳密に制御されていることが知られている 以前私たちは オートファゴソームとリソソームの融合に関わる分子として Syntaxin17 を同定し Syntaxin17-SNAP29-VAMP8 の SNARE 複合体がオートファゴソーム-リソソーム融合の重要因子であることを明らかにした しかしながら これらの SNARE 分子がどのように制御されているのか その上流因子は不明なままであった 今回私たちは LC-MS/MS 解析により Syntaxin17 と結合する分子を探索したところ HOPS 複合体の構成因子である Vps33A と Vps16 を同定した HOPS 複合体はリソソームの膜融合に関与することが報告されている tethering 複合体である そこで 私たちは HOPS 複合体が Syntaxin17 を介したオートファゴソームとリソソームの融合に関与するのかどうか 検証した HOPS 複合体の構成因子 (Vps33A, Vps39) を RNAi により発現抑制すると Syntaxin17 陽性のオートファゴソームが蓄積し オートファゴソームとリソソームの融合が阻害された 一方で これまでに HOPS 複合体と結合してオートファジーを制御すると報告されていた UVRAG は Syntaxin17 とは結合しなかった よって UVRAG はエンドサイトーシス経路を介して 間接的にオートファジーに関与していると考えられた 本研究から HOPS 複合体はエンドサイトーシスとともに Syntaxin17 と結合することで オートファゴソーム-リソソーム融合にも関与していることが明らかとなった - 5 -

6 論文審査の要旨および担当者 報告番号甲第 4700 号蒋培都 論文審査担当者 主査副査 清水重臣中田隆夫 淺原弘嗣 ( 論文審査の要旨 ) オートファジーの最終段階では 細胞質成分を包み込んだオートファゴソームとリソソームの融合が実行される この時に SNARE に関わる syntaxin 17 (STX17) 分子が必要であることは知られていたが, 両者をテザリングする分子に関しては充分な知見がなかった そこで申請者は STX17 と結合する分子を 免疫沈降 -マススペクトル解析にて探索した その結果 VPS33A, VPS16 など vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) complex に含まれる分子が同定された HOPS complex はテザリング分子として知られているものである 申請者は さらに 1HOPS complex が オートファゴソームとリソソームの融合に必要であること 2HOPS complex が機能するためには STX17 が必要であること 3HOPS complex は オートファジー制御以外に エンドソームとリソソームの融合にも関わっており この際には STX17 は不要であること を見いだした 本研究は これまで報告のなかったオートファゴソームとリソソームのテザリング因子を同定し 詳細な機能解析を行った研究であり 高く評価できる ( 1 )