酿酒酵母全基因组SNARE蛋白的亚细胞定位研究 <sup>*</sup>

doi:10.13523/j.cb.20191006

中国生物工程杂志

2019, Vol. 39

Issue (10): 44-57 DOI: 10.13523/j.cb.20191006

技术与方法

酿酒酵母全基因组SNARE蛋白的亚细胞定位研究 ^*

张正坦,朱婧,谢志平(

)

上海交通大学生命科学技术学院微生物代谢国家重点实验室上海 200240

A Subcellular Localization Survey for All SNARE Proteins in Saccharomyces cerevisiae

ZHANG Zheng-tan,ZHU Jing,XIE Zhi-ping(

)

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,Shanghai Jiao Tong University, Shanghai 200240, China

全文: PDF(4543 KB) HTML

摘要：

在真核细胞中,内质网、高尔基体、质膜等膜结构间的蛋白质运输主要通过囊泡出芽和融合实现。SNARE蛋白家族在介导囊泡与目的膜结构融合过程中发挥关键作用。在模式生物酿酒酵母中,对全基因组SNARE蛋白的系统研究仍有不足。此研究构建了一套用于标记酿酒酵母基因组全部24种SNARE的工具质粒。该系列质粒既能呈现出良好的定位特征,又避免了过度表达造成的定位异常。通过与细胞器标记共定位验证了SNARE蛋白的亚细胞定位。结果发现3种SNARE的定位与之前报道不符:Bos1定位于早高尔基体,Snc1和Bet1定位于晚高尔基体/早内体。另外,Sec9定位于芽尖和芽颈,这是首次观察到Sec9在活酵母细胞中的定位。这项工作首次全面的检验了酵母SNARE家族蛋白的亚细胞定位,为后续SNARE蛋白功能研究提供了新线索,并为相关研究提供了一套工具质粒。

关键词： SNARE; 亚细胞定位; 酿酒酵母; 共定位

Abstract:

In eukaryotic cells, protein transport between membrane structures such as endoplasmic reticulum, Golgi, and plasma membrane is mainly achieved via vesicle budding and fusion. The SNARE protein family plays a key role in mediating the fusion of vesicles with the target membrane structure. In the model organism Saccharomyces cerevisiae, systematic studies of SNARE proteins in the whole genome are still lacking. A set of plasmids for the labeling of all 24 SNAREs in S. cerevisiae with GFP are constructed. Most of the plasmids employ endogenous promoters for expression, with only a few mildly overexpressed, thus avoiding potential mislocalization caused by high overexpression. The subcellular localization of each SNARE is verified by co-localization with organelle markers. Results indicate that the localization of three SNAREs differs from those in existing literature: Bos1 localizes to early Golgi; and Snc1 and Bet1 localize to the late Golgi/early endosomes. In addition, Sec9 is detected at the bud tip and septum. This is the first time that the localization of Sec9 in vegetative cells has been observed. It is also the first comprehensive experimental evaluation of yeast SNARE subcellular localization. Furthermore, the constructed plasmids constitute a convenient tool set for future yeast cell biology studies.

Key words: SNARE Subcellular localization Saccharomyces cerevisiae Colocalization

收稿日期: 2019-03-28 出版日期: 2019-11-12

ZTFLH:

Q816

基金资助: *上海市自然科学基金探索类项目(18ZR1420400);上海市教育委员会科研创新计划(2017-01-07-00-02-E00035)

通讯作者: 谢志平 E-mail: zxie@sjtu.edu.cn

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引用本文:

张正坦,朱婧,谢志平. 酿酒酵母全基因组SNARE蛋白的亚细胞定位研究 ^*[J]. 中国生物工程杂志, 2019, 39(10): 44-57.

ZHANG Zheng-tan,ZHU Jing,XIE Zhi-ping. A Subcellular Localization Survey for All SNARE Proteins in Saccharomyces cerevisiae. China Biotechnology, 2019, 39(10): 44-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191006 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/44

Primer name			Primer sequence
Sec20 pro-F			TCCTGCAGCCCGGGGGATCCATTGTTTAATGTGGTCGTTGTAAC
Sec20 pro-R			AATTCTTCACCTTTAGACATAATCTTTAATGGCTTTATAGTTTGTT
Sec20-F			GGATCCCCGGGTTAATTAACATGGTCGTGACATTTTTGCAGGA
Sec20-R			ATACAGTTTTTTGCGGCCGCTCATAGCTCATCATGTGAGACG
Sec22 pro-F			TCCTGCAGCCCGGGGGATCCTCATAGATTTATCTTCATATGTTCT
Sec22 pro-R			AATTCTTCACCTTTAGACATCCCTACTTAATTGTGAGTGTG
Sec22-F			GGATCCCCGGGTTAATTAACATGATAAAGTCAACACTAATCTACA
Sec22-R			ATACAGTTTTTTGCGGCCGCCTATTTGAGGAAGATCCACCAG
Use1 pro-F			TCCTGCAGCCCGGGGGATCCAAGCCAAATCTGTTACGCACGG
Use1 pro-R			AATTCTTCACCTTTAGACATCTTGCCTTGCTATTATGTGTCC
Use1-F			GGATCCCCGGGTTAATTAACATGGCTGAAACTTCCAACGAC
Use1-R			ATACAGTTTTTTGCGGCCGCTTATAGGGCCGGGAATAATTGAA
Ufe1 pro-F			TCCTGCAGCCCGGGGGATCCAGCACCAAAATGCCTCCATTGA
Ufe1 pro-R			AATTCTTCACCTTTAGACATGAGTTAATAAGCTCTTCACTGC
Ufe1-F			GGATCCCCGGGTTAATTAACATGATGTCTGATTTAACACCAATAT
Ufe1-R			ATACAGTTTTTTGCGGCCGCTTAACCTACATAATCTAGGAACAAT
Sed5 pro-F			TCCTGCAGCCCGGGGGATCCCTATCGCCTCAAAGAACCACAT
Sed5 pro-R			AATTCTTCACCTTTAGACATGGGAGTTGTGTGGTATGGTGA
Sed5-F			GGATCCCCGGGTTAATTAACATGAACATAAAGGATAGAACTTCA
Sed5-R			ATACAGTTTTTTGCGGCCGCTTAATTGACTAAAACCCAAATAACG
Gos1 pro-F			TCCTGCAGCCCGGGGGATCCTGCCTGTTAGACACGAGATGTT
Gos1 pro-R			AATTCTTCACCTTTAGACATGTGGTGTGGTTGCTTGTCTGG
Gos1-F			GGATCCCCGGGTTAATTAACATGAGCTCACAACCGTCTTTCG
Primer name		Primer sequence
Gos1-R		ATACAGTTTTTTGCGGCCGCTTACCATGTGAAAAACAAAAACAGT
Bos1 pro-F		TCCTGCAGCCCGGGGGATCCACGTCTTCTATATCTGGGTTTTC
Bos1 pro-R		AATTCTTCACCTTTAGACATTCCCCCCTCAAATCAATATGATT
Bos1-F		GGATCCCCGGGTTAATTAACATGGTATGTTTGATCGCCGGA
Bos1-R		ATACAGTTTTTTGCGGCCGCCTATCTTAACCATTTCAACACATAA
Sft1 pro-F		TCCTGCAGCCCGGGGGATCCGGCGGAAAGTTAGGCCGACA
Sft1 pro-R		AATTCTTCACCTTTAGACATCGCTATGATGATTATATATTACCTT
Sft1-F		GGATCCCCGGGTTAATTAACATGTCAAATTCTAGGTATTCTCAG
Sft1-R		ATACAGTTTTTTGCGGCCGCCTAAAATAACTTAAACAGGGTATATA
Tlg1 pro-F		TCCTGCAGCCCGGGGGATCCGATAGTCCCCCATTTTTTTTTATGT
Tlg1 pro-R		AATTCTTCACCTTTAGACATTTGTTAAAGAAAGGATCTTAGCAAT
Tlg1-F		GGATCCCCGGGTTAATTAACATGAACAACAGTGAAGATCCGTT
Tlg1-R		ATACAGTTTTTTGCGGCCGCTCAAGCAATGAATGCCAAAACTAA
Tlg2 pro-F		TCCTGCAGCCCGGGGGATCCCACCCATTTAAATTTGACGATAAA
Tlg2 pro-R		AATTCTTCACCTTTAGACATGTTTGTAACGACTGCCTAGATC
Tlg2-F		GGATCCCCGGGTTAATTAACATGTTTAGAGATAGAACTAATTTATTT
Tlg2-R		ATACAGTTTTTTGCGGCCGCTCAAAGTAGGTCATCCAAAGCAT
Snc1 pro-F		TCCTGCAGCCCGGGGGATCCTAATATGTCCTTAGAATGGGGAA
Snc1 pro-R		AATTCTTCACCTTTAGACATGGACAATGAATACGTTGCGCTT
Snc1-F		GGATCCCCGGGTTAATTAACATGTCGTCATCTACTCCCTTTGA
Snc1-R		ATACAGTTTTTTGCGGCCGCCTATCGACTAAAGTGAACAGCAA
Snc2 pro-F		TCCTGCAGCCCGGGGGATCCGTGTTTCGTTAGGTGGGCCG
Snc2 pro-R		AATTCTTCACCTTTAGACATCGTTGCGCGTTCTTATTTTGTA
Snc2-F		GGATCCCCGGGTTAATTAACATGTCGTCATCAGTGCCATACG
Snc2-R		ATACAGTTTTTTGCGGCCGCTTAGCTGAAATGGACGACGATAG
Bet1 pro-F		TCCTGCAGCCCGGGGGATCCCCAGGCGACGTCAGAGATGA
Bet1 pro-R		AATTCTTCACCTTTAGACATCTGTGTAGCCTAGTGTTGTGA
Bet1-F		GGATCCCCGGGTTAATTAACATGAGTTCAAGGTATGAGAAATTAT
Bet1-R		ATACAGTTTTTTGCGGCCGCTTATGTAATCCATACCCAAAAAAATA
Vam3 pro-F		TCCTGCAGCCCGGGGGATCCGAAAACAAGGAAAAAACAACAGAA
Vam3 pro-R		AATTCTTCACCTTTAGACATAATCTCAACTTTCTGCAGTGGAT
Vam3-F		GGATCCCCGGGTTAATTAACATGTCCTTTTTCGACATCGAAG
Vam3-R		ATACAGTTTTTTGCGGCCGCCTAACTTAATACAGCAAGCAATAC
Vam7 pro-F		TCCTGCAGCCCGGGGGATCCGCCTATTCTTTCACCAAATTCAG
Vam7 pro-R		AATTCTTCACCTTTAGACATCAATATCAATTATCAACCCTTATATG
Vam7-F		GGATCCCCGGGTTAATTAACATGGCAGCTAATTCTGTAGGGA
Vam7-R		ATACAGTTTTTTGCGGCCGCTCAAGCACTGTTGTTAAAATGTCT
Nyv1 pro-F		TCCTGCAGCCCGGGGGATCCAAGGCGGCAAGACTTCAAGAAG
Nyv1 pro-R		AATTCTTCACCTTTAGACATTTGGTAAAAATATAAAAGTCCAAT
Nyv1-F		GGATCCCCGGGTTAATTAACATGAAACGCTTTAATGGTATGTATA
Nyv1-R		ATACAGTTTTTTGCGGCCGCTTACCACAGATAGAAAAACATGAAA
Primer name	Primer sequence
Vti1 pro-F	TCCTGCAGCCCGGGGGATCCATTGATTATCCTTCAAGTCCATC
Vti1 pro-R	AATTCTTCACCTTTAGACATAGTAAGCCATGCAGCAATGTAAA
Vti1-F	GGATCCCCGGGTTAATTAACATGAGTTCCCTATTAATATCATACG
Vti1-R	ATACAGTTTTTTGCGGCCGCTTATTTAAACTTTGAGAACAAAACTA
Pep12 pro-F	TCCTGCAGCCCGGGGGATCCTACTTAGTCCTTTGGTCAAAAATAT
Pep12 pro-R	AATTCTTCACCTTTAGACATCTCAACACAATTATTGTAGTAATTTA
Pep12-F	GGATCCCCGGGTTAATTAACATGTCGGAAGACGAATTTTTTGG
Pep12-R	ATACAGTTTTTTGCGGCCGCTTACAATTTCATAATGAGAAAAATAAAA
Syn8 pro-F	TCCTGCAGCCCGGGGGATCCTGAAAGCTCGTACTTCTGTCGC
Syn8 pro-R	AATTCTTCACCTTTAGACATACCGTGTGTGCTTACGCATCTA
Syn8-F	GGATCCCCGGGTTAATTAACATGGATGTGTTGAAGCTGGGTT
Syn8-R	ATACAGTTTTTTGCGGCCGCTTATAATACTAATAGAAGCAACAGG
Ykt6 pro-F	TCCTGCAGCCCGGGGGATCCTTCTTCTTCTCCATTATTATCTTCA
Ykt6 pro-R	AATTCTTCACCTTTAGACATTGCCAAAATAACTTCTCTAGTGAT
Ykt6-F	GGATCCCCGGGTTAATTAACATGAGAATCTACTACATCGGTG
Ykt6-R	ATACAGTTTTTTGCGGCCGCCTACATGATGATGCAACACGAAT
Sso1 pro-F	TCCTGCAGCCCGGGGGATCCTACCGAAGAAAGAATACGACGAT
Sso1 pro-R	AATTCTTCACCTTTAGACATTTGATTTGTTTCTATTTTTAATTGCC
Sso1-F	GGATCCCCGGGTTAATTAACATGAGTTATAATAATCCGTACCAG
Sso1-R	ATACAGTTTTTTGCGGCCGCTTAACGCGTTTTGACAACGGCT
Sso2 pro-F	TCCTGCAGCCCGGGGGATCCTATAAATATAATAATATTATTGATTAATTA
Sso2 pro-R	AATTCTTCACCTTTAGACATTGCTGCAATATTTGTGCGTGTAT
Sso2-F	GGATCCCCGGGTTAATTAACATGAGCAACGCTAATCCTTATGA
Sso2-R	ATACAGTTTTTTGCGGCCGCTTACTTTCTTGTTTCCACAACGG
Sec9 pro-F	TCCTGCAGCCCGGGGGATCCTCTCTCTCTCTCTCTACTTAACA
Sec9 pro-R	AATTCTTCACCTTTAGACATTCTTTTGGTGTCAATGGTGTATTA
Sec9-F	GGATCCCCGGGTTAATTAACATGGGATTAAAGAAATTTTTTAAGAT
Sec9-R	ATACAGTTTTTTGCGGCCGCCTATCTGATACCTGCCAACCTG
Spo20 pro-F	TCCTGCAGCCCGGGGGATCCAAAAAGCCGTTTGCGGAAAGCA
Spo20 pro-R	AATTCTTCACCTTTAGACATTATATATCTAAAAATGGCTATTCACA
Spo20-F	GGATCCCCGGGTTAATTAACATGGGGTTCAGAAAAATACTTGCTA
Spo20-R	ATACAGTTTTTTGCGGCCGCTCACCATCTTTTCCCGATCACT
BS-Ura-F	CCTTTTTTGCGAGGCATATTTATG
BS-Ura-R	GTAACTATTGAATTTTGTTTGGATTT
GFP-F	ATGTCTAAAGGTGAAGAATTATTCA
GFP-Fa6a-R	GTTAATTAACCCGGGGATCCGTCGACCTGCAGCGTACGAAGCTTTGTACAATTCATCCATACCATG
Stu1-F	CCAATTGAGGCCTTTTTTGCGAGGCATATTTAT
Stu1-R	AAAAAGGCCTCAATTGGCGCAGTAGCCTCA
Snab1-F	TAGTTACGTAGACGTCCTACGATTCCGCGG
Snab1-R	GGACGTCTACGTAACTATTGAATTTTGTTTGGATT

表1 引物及DNA序列

表2 质粒及线性化所用的酶切位点

图1 GFP-SNARE系列质粒结构示意图

图2 呈现胞内点状分布的SNARE

图3 定位于较大细胞器的SNARE

图4 位于质膜SNARE

图5 定位在内质网或细胞核的SNARE

图6 定位在早高尔基体的SNARE

图7 定位在晚高尔基体/早内体的SNARE

图8 定位在液泡或晚内体的SNARE

表3 酿酒酵母全基因组SNARE蛋白的亚细胞定位

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