酿酒酵母中TRAPP复合体专一性亚基在囊泡运输和细胞自噬中的功能研究
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摘要
囊泡运输与细胞自噬是真核生物维持正常生命活动所必需的生理过程,其中囊泡运输是实现细胞内物质运输的重要途径,而细胞自噬则是维持细胞内生理平衡并帮助细胞度过逆境的一种细胞内物质降解途径。
酿酒酵母(Saccharomyces cerevisiae)细胞中不同细胞器之间的囊泡运输受到Ypt/Rab家族小G蛋白(GTPases)的调控,而小G蛋白又受到鸟苷酸交换因子(GEFs)和GTPase激活蛋白(GAPs)的调节,使小G蛋白在GTP结合态(活性形态)和GDP结合态(非活性形态)之间循环。酿酒酵母中的TRAPP (Transport Protein Particle, TRAPP)复合体是多亚基大分子复合体,可作为GEFs激活小G蛋白Yptl和Ypt31/32。到目前为止,研究认为TRAPP复合体在细胞内存在三种类型,其中TRAPPⅡ复合体可以激活Yptl,调控着内质网到高尔基体的囊泡运输；TRAPPⅡ复合体可以激活Ypt31/32,调控着高尔基体到质膜及内涵体到高尔基体的囊泡运输；TRAPPⅢ复合体同样可激活Ypt1,但参与调控细胞自噬过程。Trs85是TRAPPⅢ复合体专一性亚基,在细胞自噬途径中发挥重要作用,但也有实验显示过量表达Trs85能恢复TRAPP复合体亚基Bet3和Bet5突变体在高温下生长,另有实验发现在GFP标记v-SNARE蛋白Snc1的菌株中敲除TRS85将导致GFP-Snc1的运输受阻,且表型与TRAPPⅡ复合体专一性亚基突变类似。这些遗传学上的实验证据表明Trs85也参与囊泡运输,但具体机制并不清楚。Trs130是TRAPPⅡ复合体的专一性亚基,大量实验数据表明,Trs130通过其所在的TRAPP复合体作为GEF调控Ypt31/32,参与囊泡运输,但并不清楚Trs130及其他TRAPPⅡ专一性亚基是否参与细胞自噬过程。近年发现小G蛋白Ypt31/32调控细胞自噬过程,那么作为GEF调控Ypt31/32的TRAPPⅡ复合体是否会调控细胞自噬过程?为了全面探讨含有专一性亚基的TRAPPⅢ和TRAPPⅡ复合体分别在囊泡运输与细胞自噬中的作用及机制,本研究在已有报道Trs85调控细胞自噬和Trs130调控囊泡运输的基础上,着重剖析Trs85在囊泡运输和Trs130在细胞自噬中的功能及作用机制,得到如下几方面的主要结果：
1.Trs85调控细胞内内质网到高尔基体的物质运输
在GFP标记Snc1蛋白的菌株中敲除TRS85,通过跟踪GFP-Snc1的运输来反映细胞内囊泡运输的情况。结果发现：(1)在GFP标记Sncl的菌株中敲除TRS85,引起菌株对高温敏感,计为trs85Δts。(2)高温处理该菌株后观察荧光,发现trs85Δts中GFP-Snc1不能被正常地运输到质膜上,而是积累在内质网,与ypt1ts中所出现的GFP-Snc1表型类似。这两种突变体在高温下所出现的GFP-Snc1表型不同于对应的另外两种突变体trs130ts和ypt31Δ/32ts中的GFP-Snc1表型。后两种突变体在高温下GFP-Sncl也不能被正常运输到质膜上,但积累在内涵体上。这些结果表明,Trs85与Yptl调控着囊泡从内质网到高尔基体的运输,而Trs130与Ypt31/32则调控着囊泡从内涵体到高尔基体的运输。
2.过量表达Ypt1和Ypt31/32能分别恢复trs85Δts和trs130ts突变体的生长及囊泡运输缺陷
GFP-Snc1在Trs85和Yptl的突变体中存在着相似的荧光表型。为了探讨Trs85及其所在的TRAPPⅢ复合体是否通过Yptl调控囊泡运输,本研究在trs85Δts和trs130ts突变体中过量表达Yptl和Ypt31/32,结果显示：过量表达Yptl能恢复trs85Δts突变体在高温下生长及解除GFP-Snc1运输受阻,而过量表达Ypt31/32则没有类似效果；相对应地,过量表达Ypt31/32能恢复trs130ts突变体在高温下生长及解除GFP-Snc1运输受阻,而过量表达Yptl则没有类似效果。这些结果表明,Trs85或其所在的TRAPPⅢ复合体通过调控Yptl参与内质网到高尔基体的囊泡运输,Trs130则是通过调控Ypt31/32参与内涵体到高尔基体的囊泡运输。
3.Trs130调控细胞自噬过程中Atg8和Atg9从反式高尔基体向PAS的募集
Pho8△60碱性磷酸酶的活性水平是检测细胞自噬是否正常的重要指标。本研究在TN124为背景的菌株中构建trs130ts突变体检测Pho8A60活性,发现在限制性温度下诱导细胞自噬,突变体中碱性磷酸酶的活性远低于野生型。通过Western blot定量分析,检测营养丰富或氮饥饿诱导细胞自噬的条件下prApel成熟与Atg8降解的情况,结果显示：在限制性温度下,trs130ts突变体中prApel在细胞内的成熟及Atg8在细胞内的降解受到阻碍。通过荧光观察GFP-Atg8的定位跟踪细胞内自噬过程,发现限制性温度下trs130ts突变体中GFP-Atg8不能被运输到液泡,并在细胞内积累形成多个绿色亮点。Atg9被认为是细胞自噬相关蛋白中唯一一个跨膜蛋白,Atg8的运输依赖于Atg9的功能。通过敲除ATG1后荧光观察Atg9的定位,发现限制性温度下trs130ts突变体中Atg9往PAS位点的顺向运输同样受阻。以上结果都表明trs130ts突变体中细胞自噬过程受阻。
细胞自噬相关蛋白Atg11和Atg17作为脚手架蛋白,招募其它细胞自噬相关蛋白至PAS位点。通过敲除ATG11和ATG17发现,GFP-Atg8在trs130ts突变体细胞内依然会积累成多个亮点,表型与基因敲除前相似,说明Trs130作用于Atg8被招募至PAS位点之前。Sec7是分泌过程中的重要蛋白,通过Sec7-DsRed定位反式高尔基体来分析GFP-Atg8和Atg9-GFP在反式高尔基体上的分布情况,发现限制性温度下trs130ts突变体中一部分的Atg8和Atg9蛋白会与Sec7重合,不同于野生型菌株中很少有积累的情况,说明Trs130调控Atg8和Atg9从反式高尔基体到PAS的募集。
4.Trs130通过Ypt31/32的活性介导细胞白噬
至此,Trs130和Ypt31/32都参与细胞自噬过程。已知Trs130通过调控Ypt31/32的活性介导囊泡运输过程,但不清楚在细胞自噬过程中两者之间是否仍然存在类似关系?本研究发现,Trs130突变所造成的自噬缺陷表型可以通过过量表达小G蛋白Ypt32、Ypt31及GTP结合态的Ypt31得到恢复,而过量表达Yptl和GDP结合态的Ypt31则不能恢复。根据这一结果可以得出,细胞自噬中Trs130通过影响Ypt31/32的活性介导着细胞自噬过程,说明Trs130与Ypt31/32之间的GEF与GTPaes的对应关系同时存在于囊泡运输与细胞自噬过程中。
本论文研究了酿酒酵母中TRAPP复合体专一性亚基Trs85和Trs130分别参与囊泡运输和细胞自噬的过程,研究结果为全面阐述TRAPPⅢ和TRAPPⅡ复合体及其专一性亚基在囊泡运输与细胞自噬这两大生理过程中的功能及作用机制提供了充分的实验依据,为进一步研究不同类型TRAPP复合体之间的联系提供了坚实的理论基础。
Vesicle trafficking and autophagy are basic physiological processes in eukaryotes for cell survival. Vesicle trafficking is an important way for the transport of substances within the cell, while autophagy is an essential cellular degradation process that eliminates obsoletes or damaged cytoplasmic materials to maintain intracellular homeostasis.
Vesicle transport among different organelles in yeast is regulated by a molecular switch named Ypt/Rab GTPases, which are activated by Guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs) to cycle between GTP-bound form and GDP-bound form. TRAPP (Transport protein particle) complex is one of the large multisubunit complexes and is implicated in tethering vesicles. Three TRAPP complexes (Ⅰ, Ⅱ and Ⅲ) are reported in yeast for their GEF activities for GTPases Yptl and Ypt31/32. TRAPPI regulates endoplasmic reticulum(ER)-to-Golgi trafficking by activating Ypt1, TRAPPII regulates Golgi-to-plasma membrane and endosome-to-Golgi trafficking by activating Ypt31/32. TRAPPIII regulates autophagy by activating Yptl Trs85is a specific subunit of TRAPPIII complex which has important roles in autophagy. There are also data showing that the overexpression of Trs85can suppress the growth defect of bet3ts and bet5ts (Bet3and Bet5is the subunit of TRAPP complex) mutants at high temperature. Others further reported that the GFP-Sncl (Sncl is a v-SNARE protein) transport was blocked in trs85A mutant and the phenotype is similar to the mutant of TRAPPII-specific subunits. These genetic evidences indicate that Trs85participates in vesicle trafficking with unknown mechanism. Trs130is a specific subunit of TRAPPII complex, and some data suggest that Trs130is involved in the transport of substance to exit the Golgi. It is unknown whether Trs130and other TRAPPⅡ-specific subunits regulate autophagy. Recently, Ypt31/32was shown to regulate autophagy. It was speculated that TRAPPII complex may regulate autophagy through its substrate Ypt31/32. In order to globally elucidate the functions of TRAPPIII and TRAPPII (both with specific TRAPP subunits) in both vesicle trafficking and autophagy, this study focuses on the functions and mechanisms of Trs85in vesicle trafficking and Trsl30in autophagy. The main results are listed below:
1. Trs85played a role in ER-to-Golgi vesicle trafficking in yeast
GFP-Snc1was used as a probe to reflect the process of vesicle trafficking in yeast. Results found are:(1) GFP-Sncl tagged strains became temperature-sensitive when TRS85was deleted, the resulting strain designated as trs85Δts;(2) GFP-Sncl accumulated in the ER and did not recycle to the PM in trs85Δts mutant cells after incubated at high temperature treatment. Similar GFP-Sncl phenotype was found in yptlts mutant at the restrictive temperature. This internal GFP-Sncl phenotypes in both mutants were different from those in ypt31A/32ts and trs130ts mutant cells, in which GFP-Sncl accumulated in endosomes with defect to reach the PM at the restrictive temperature. Results suggest that Trs85and Yptl regulate ER-to-Golgi transport; whereas Trs130and Ypt31/32regulate endosome-to-Golgi transport.
2. Grouping Yptl with Trs85and Ypt31/32with Trs130in vesicle trafficking
There are similar GFP-Sncl phenotypes between trs85Δts and yptlts mutant. In order to know whether Trs85functions through Yptl in vesicular transport, Yptl and Ypt31/32were overexpressed in trs85Δts and trs130ts mutant cells. Results are:Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs85Δts mutant cells at the restrictive temperature, while Ypt31/32did not. In contrast, Ypt31/32, but not Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs130ts mutant cells at the restrictive temperature. These results suggest that Trs85or TRAPPIII regulates ER-to-Golgi transport through Yptl while Trs130regulates endosome-to-Golgi transport through Ypt31/32.
3. Trs130regulated the recruitment of Atg8and Atg9from the trans-Golgi to the PAS in autophagy.
The Pho8A60assay is a quantitative method to detect autophagy. When autophagy was induced at a non-permissive temperature (NPT), the Pho8Δ60activity in trs130ts mutant is much lower than that in wild-type. Immunoblot assay was further applied to determine GFP-Atg8degradation and prApe1maturation in the trs130ts mutant in the process of autophagy. Results showed that prApel maturation and GFP-Atg8degradation were impaired in trs130ts cells under both rich and starvation conditions at NPT. By fluorescence microscopy, GFP-Atg8was found to accumulate in the cytosol as multiple dots at NPT. The Atg9is the only transmembrane protein among Atg proteins and Atg8transport depends on Atg9. Using TAKA assay, we found that Atg9anterograde transport was defective in trs130ts mutant at NPT independent of culture medium. These results indicate that autophagy processes are blocked in trs130ts mutants.
Atg-related protein Atg11and Atg17are scaffolding proteins, and both can recruit other Atg proteins to the PAS in the Cvt pathway and autophagy. GFP-Atg8in trs130ts mutant still accumulated as multiple dots in the absence of ATG11and ATG17. This indicates Trsl30functions upstream of the recruitment of Atg proteins to the PAS. Sec7is indispensable for secretion and localizes to the trans-Golgi. Using Sec7-DeRed as a trans-Golgi marker, Atg8and Atg9were found to be partially trapped in the trans-Golgi, not like the few localization of Atg8and Atg9in the trans-Golgi in wild-type. In summary, autophagy was impaired in trs130ts mutant and Trs130regulates the recruitment of Atg8and Atg9from the trans-Golgi to the PAS.
4. Trs130mediates autophagy through the activity of Yptt31/32
Untill now, both Trs130and Ypt31/32regulate autophagy. Trs130mediates vesicle trafficking through Ypt31/32, but it is unclear whether Trs130mediates autophagy through Ypt31/32. In this study, we found that the overexpression of Ypt32, Ypt31or the GTP-bound form of Ypt31, but not Yptl or the GTP-bound form of Ypt31, rescued autophagy defects in trs130ts mutant cells. We conclude that Trs130participates in autophagy through the activity of Ypt31/32and suggest that the GEF-GTPases relationship existing in both vesicle trafficking and autophagy.
This study elucidate the functions and mechanisms of Trs85and Trs130in vesicle trafficking and autophagy respectively. The results obtained here provide comprehensive experimental evidence for the roles and mechanisms of TRAPPIII and TRAPPⅡ in vesicle trafficking and autophagy, which will facilate the studies of the functional relationships among TRAPP complexes.
酿酒酵母(Saccharomyces cerevisiae)细胞中不同细胞器之间的囊泡运输受到Ypt/Rab家族小G蛋白(GTPases)的调控,而小G蛋白又受到鸟苷酸交换因子(GEFs)和GTPase激活蛋白(GAPs)的调节,使小G蛋白在GTP结合态(活性形态)和GDP结合态(非活性形态)之间循环。酿酒酵母中的TRAPP (Transport Protein Particle, TRAPP)复合体是多亚基大分子复合体,可作为GEFs激活小G蛋白Yptl和Ypt31/32。到目前为止,研究认为TRAPP复合体在细胞内存在三种类型,其中TRAPPⅡ复合体可以激活Yptl,调控着内质网到高尔基体的囊泡运输；TRAPPⅡ复合体可以激活Ypt31/32,调控着高尔基体到质膜及内涵体到高尔基体的囊泡运输；TRAPPⅢ复合体同样可激活Ypt1,但参与调控细胞自噬过程。Trs85是TRAPPⅢ复合体专一性亚基,在细胞自噬途径中发挥重要作用,但也有实验显示过量表达Trs85能恢复TRAPP复合体亚基Bet3和Bet5突变体在高温下生长,另有实验发现在GFP标记v-SNARE蛋白Snc1的菌株中敲除TRS85将导致GFP-Snc1的运输受阻,且表型与TRAPPⅡ复合体专一性亚基突变类似。这些遗传学上的实验证据表明Trs85也参与囊泡运输,但具体机制并不清楚。Trs130是TRAPPⅡ复合体的专一性亚基,大量实验数据表明,Trs130通过其所在的TRAPP复合体作为GEF调控Ypt31/32,参与囊泡运输,但并不清楚Trs130及其他TRAPPⅡ专一性亚基是否参与细胞自噬过程。近年发现小G蛋白Ypt31/32调控细胞自噬过程,那么作为GEF调控Ypt31/32的TRAPPⅡ复合体是否会调控细胞自噬过程?为了全面探讨含有专一性亚基的TRAPPⅢ和TRAPPⅡ复合体分别在囊泡运输与细胞自噬中的作用及机制,本研究在已有报道Trs85调控细胞自噬和Trs130调控囊泡运输的基础上,着重剖析Trs85在囊泡运输和Trs130在细胞自噬中的功能及作用机制,得到如下几方面的主要结果：
1.Trs85调控细胞内内质网到高尔基体的物质运输
在GFP标记Snc1蛋白的菌株中敲除TRS85,通过跟踪GFP-Snc1的运输来反映细胞内囊泡运输的情况。结果发现：(1)在GFP标记Sncl的菌株中敲除TRS85,引起菌株对高温敏感,计为trs85Δts。(2)高温处理该菌株后观察荧光,发现trs85Δts中GFP-Snc1不能被正常地运输到质膜上,而是积累在内质网,与ypt1ts中所出现的GFP-Snc1表型类似。这两种突变体在高温下所出现的GFP-Snc1表型不同于对应的另外两种突变体trs130ts和ypt31Δ/32ts中的GFP-Snc1表型。后两种突变体在高温下GFP-Sncl也不能被正常运输到质膜上,但积累在内涵体上。这些结果表明,Trs85与Yptl调控着囊泡从内质网到高尔基体的运输,而Trs130与Ypt31/32则调控着囊泡从内涵体到高尔基体的运输。
2.过量表达Ypt1和Ypt31/32能分别恢复trs85Δts和trs130ts突变体的生长及囊泡运输缺陷
GFP-Snc1在Trs85和Yptl的突变体中存在着相似的荧光表型。为了探讨Trs85及其所在的TRAPPⅢ复合体是否通过Yptl调控囊泡运输,本研究在trs85Δts和trs130ts突变体中过量表达Yptl和Ypt31/32,结果显示：过量表达Yptl能恢复trs85Δts突变体在高温下生长及解除GFP-Snc1运输受阻,而过量表达Ypt31/32则没有类似效果；相对应地,过量表达Ypt31/32能恢复trs130ts突变体在高温下生长及解除GFP-Snc1运输受阻,而过量表达Yptl则没有类似效果。这些结果表明,Trs85或其所在的TRAPPⅢ复合体通过调控Yptl参与内质网到高尔基体的囊泡运输,Trs130则是通过调控Ypt31/32参与内涵体到高尔基体的囊泡运输。
3.Trs130调控细胞自噬过程中Atg8和Atg9从反式高尔基体向PAS的募集
Pho8△60碱性磷酸酶的活性水平是检测细胞自噬是否正常的重要指标。本研究在TN124为背景的菌株中构建trs130ts突变体检测Pho8A60活性,发现在限制性温度下诱导细胞自噬,突变体中碱性磷酸酶的活性远低于野生型。通过Western blot定量分析,检测营养丰富或氮饥饿诱导细胞自噬的条件下prApel成熟与Atg8降解的情况,结果显示：在限制性温度下,trs130ts突变体中prApel在细胞内的成熟及Atg8在细胞内的降解受到阻碍。通过荧光观察GFP-Atg8的定位跟踪细胞内自噬过程,发现限制性温度下trs130ts突变体中GFP-Atg8不能被运输到液泡,并在细胞内积累形成多个绿色亮点。Atg9被认为是细胞自噬相关蛋白中唯一一个跨膜蛋白,Atg8的运输依赖于Atg9的功能。通过敲除ATG1后荧光观察Atg9的定位,发现限制性温度下trs130ts突变体中Atg9往PAS位点的顺向运输同样受阻。以上结果都表明trs130ts突变体中细胞自噬过程受阻。
细胞自噬相关蛋白Atg11和Atg17作为脚手架蛋白,招募其它细胞自噬相关蛋白至PAS位点。通过敲除ATG11和ATG17发现,GFP-Atg8在trs130ts突变体细胞内依然会积累成多个亮点,表型与基因敲除前相似,说明Trs130作用于Atg8被招募至PAS位点之前。Sec7是分泌过程中的重要蛋白,通过Sec7-DsRed定位反式高尔基体来分析GFP-Atg8和Atg9-GFP在反式高尔基体上的分布情况,发现限制性温度下trs130ts突变体中一部分的Atg8和Atg9蛋白会与Sec7重合,不同于野生型菌株中很少有积累的情况,说明Trs130调控Atg8和Atg9从反式高尔基体到PAS的募集。
4.Trs130通过Ypt31/32的活性介导细胞白噬
至此,Trs130和Ypt31/32都参与细胞自噬过程。已知Trs130通过调控Ypt31/32的活性介导囊泡运输过程,但不清楚在细胞自噬过程中两者之间是否仍然存在类似关系?本研究发现,Trs130突变所造成的自噬缺陷表型可以通过过量表达小G蛋白Ypt32、Ypt31及GTP结合态的Ypt31得到恢复,而过量表达Yptl和GDP结合态的Ypt31则不能恢复。根据这一结果可以得出,细胞自噬中Trs130通过影响Ypt31/32的活性介导着细胞自噬过程,说明Trs130与Ypt31/32之间的GEF与GTPaes的对应关系同时存在于囊泡运输与细胞自噬过程中。
本论文研究了酿酒酵母中TRAPP复合体专一性亚基Trs85和Trs130分别参与囊泡运输和细胞自噬的过程,研究结果为全面阐述TRAPPⅢ和TRAPPⅡ复合体及其专一性亚基在囊泡运输与细胞自噬这两大生理过程中的功能及作用机制提供了充分的实验依据,为进一步研究不同类型TRAPP复合体之间的联系提供了坚实的理论基础。
Vesicle trafficking and autophagy are basic physiological processes in eukaryotes for cell survival. Vesicle trafficking is an important way for the transport of substances within the cell, while autophagy is an essential cellular degradation process that eliminates obsoletes or damaged cytoplasmic materials to maintain intracellular homeostasis.
Vesicle transport among different organelles in yeast is regulated by a molecular switch named Ypt/Rab GTPases, which are activated by Guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs) to cycle between GTP-bound form and GDP-bound form. TRAPP (Transport protein particle) complex is one of the large multisubunit complexes and is implicated in tethering vesicles. Three TRAPP complexes (Ⅰ, Ⅱ and Ⅲ) are reported in yeast for their GEF activities for GTPases Yptl and Ypt31/32. TRAPPI regulates endoplasmic reticulum(ER)-to-Golgi trafficking by activating Ypt1, TRAPPII regulates Golgi-to-plasma membrane and endosome-to-Golgi trafficking by activating Ypt31/32. TRAPPIII regulates autophagy by activating Yptl Trs85is a specific subunit of TRAPPIII complex which has important roles in autophagy. There are also data showing that the overexpression of Trs85can suppress the growth defect of bet3ts and bet5ts (Bet3and Bet5is the subunit of TRAPP complex) mutants at high temperature. Others further reported that the GFP-Sncl (Sncl is a v-SNARE protein) transport was blocked in trs85A mutant and the phenotype is similar to the mutant of TRAPPII-specific subunits. These genetic evidences indicate that Trs85participates in vesicle trafficking with unknown mechanism. Trs130is a specific subunit of TRAPPII complex, and some data suggest that Trs130is involved in the transport of substance to exit the Golgi. It is unknown whether Trs130and other TRAPPⅡ-specific subunits regulate autophagy. Recently, Ypt31/32was shown to regulate autophagy. It was speculated that TRAPPII complex may regulate autophagy through its substrate Ypt31/32. In order to globally elucidate the functions of TRAPPIII and TRAPPII (both with specific TRAPP subunits) in both vesicle trafficking and autophagy, this study focuses on the functions and mechanisms of Trs85in vesicle trafficking and Trsl30in autophagy. The main results are listed below:
1. Trs85played a role in ER-to-Golgi vesicle trafficking in yeast
GFP-Snc1was used as a probe to reflect the process of vesicle trafficking in yeast. Results found are:(1) GFP-Sncl tagged strains became temperature-sensitive when TRS85was deleted, the resulting strain designated as trs85Δts;(2) GFP-Sncl accumulated in the ER and did not recycle to the PM in trs85Δts mutant cells after incubated at high temperature treatment. Similar GFP-Sncl phenotype was found in yptlts mutant at the restrictive temperature. This internal GFP-Sncl phenotypes in both mutants were different from those in ypt31A/32ts and trs130ts mutant cells, in which GFP-Sncl accumulated in endosomes with defect to reach the PM at the restrictive temperature. Results suggest that Trs85and Yptl regulate ER-to-Golgi transport; whereas Trs130and Ypt31/32regulate endosome-to-Golgi transport.
2. Grouping Yptl with Trs85and Ypt31/32with Trs130in vesicle trafficking
There are similar GFP-Sncl phenotypes between trs85Δts and yptlts mutant. In order to know whether Trs85functions through Yptl in vesicular transport, Yptl and Ypt31/32were overexpressed in trs85Δts and trs130ts mutant cells. Results are:Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs85Δts mutant cells at the restrictive temperature, while Ypt31/32did not. In contrast, Ypt31/32, but not Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs130ts mutant cells at the restrictive temperature. These results suggest that Trs85or TRAPPIII regulates ER-to-Golgi transport through Yptl while Trs130regulates endosome-to-Golgi transport through Ypt31/32.
3. Trs130regulated the recruitment of Atg8and Atg9from the trans-Golgi to the PAS in autophagy.
The Pho8A60assay is a quantitative method to detect autophagy. When autophagy was induced at a non-permissive temperature (NPT), the Pho8Δ60activity in trs130ts mutant is much lower than that in wild-type. Immunoblot assay was further applied to determine GFP-Atg8degradation and prApe1maturation in the trs130ts mutant in the process of autophagy. Results showed that prApel maturation and GFP-Atg8degradation were impaired in trs130ts cells under both rich and starvation conditions at NPT. By fluorescence microscopy, GFP-Atg8was found to accumulate in the cytosol as multiple dots at NPT. The Atg9is the only transmembrane protein among Atg proteins and Atg8transport depends on Atg9. Using TAKA assay, we found that Atg9anterograde transport was defective in trs130ts mutant at NPT independent of culture medium. These results indicate that autophagy processes are blocked in trs130ts mutants.
Atg-related protein Atg11and Atg17are scaffolding proteins, and both can recruit other Atg proteins to the PAS in the Cvt pathway and autophagy. GFP-Atg8in trs130ts mutant still accumulated as multiple dots in the absence of ATG11and ATG17. This indicates Trsl30functions upstream of the recruitment of Atg proteins to the PAS. Sec7is indispensable for secretion and localizes to the trans-Golgi. Using Sec7-DeRed as a trans-Golgi marker, Atg8and Atg9were found to be partially trapped in the trans-Golgi, not like the few localization of Atg8and Atg9in the trans-Golgi in wild-type. In summary, autophagy was impaired in trs130ts mutant and Trs130regulates the recruitment of Atg8and Atg9from the trans-Golgi to the PAS.
4. Trs130mediates autophagy through the activity of Yptt31/32
Untill now, both Trs130and Ypt31/32regulate autophagy. Trs130mediates vesicle trafficking through Ypt31/32, but it is unclear whether Trs130mediates autophagy through Ypt31/32. In this study, we found that the overexpression of Ypt32, Ypt31or the GTP-bound form of Ypt31, but not Yptl or the GTP-bound form of Ypt31, rescued autophagy defects in trs130ts mutant cells. We conclude that Trs130participates in autophagy through the activity of Ypt31/32and suggest that the GEF-GTPases relationship existing in both vesicle trafficking and autophagy.
This study elucidate the functions and mechanisms of Trs85and Trs130in vesicle trafficking and autophagy respectively. The results obtained here provide comprehensive experimental evidence for the roles and mechanisms of TRAPPIII and TRAPPⅡ in vesicle trafficking and autophagy, which will facilate the studies of the functional relationships among TRAPP complexes.
引文
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