Sec12 binds to Sec16 at transitional ER sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12-Sec16 interaction has a conserved role in ER export.     

De novo formation of transitional ER sites and Golgi structures in Pichia pastoris

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.     

Sec16 is a determinant of transitional ER organization

BACKGROUND: Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization. RESULTS: We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks. CONCLUSIONS: Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization.     

The budding yeast Pichia pastoris has a novel Sec23p homolog

In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non-essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non-essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two-hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.     

Sec16 influences transitional ER sites by regulating rather than organizing COPII

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.     

人白细胞介素12基因在巴斯德毕赤酵母细胞中的表达(英文)

人白细胞介素 12 (hIL 12 )是人体内具有多种生物学活性的免疫调节因子 ,由p4 0和p35两个亚基经多对二硫键连接而成 .根据hIL 12的结构特点 ,采用LiCl二次转化将hIL 12的p4 0和p35两亚基基因导入巴斯德毕赤酵母X33细胞中 ,并经同源交换分别插入酵母基因组AOX1区域 ,构建成含hIL 12双亚基基因的酵母工程菌PichiapastorisX33 p4 0 p35 .经 0 .5 %甲醇诱导 ,p4 0和p35两亚基在同一酵母细胞中得到了表达 ,并组装成具有生物学活性的hIL 12p70分子     

Two mammalian Sec16 homologues have nonredundant functions in endoplasmic reticulum (ER) export and transitional ER organization

Budding yeast Sec16 is a large peripheral endoplasmic reticulum (ER) membrane protein that functions in generating COPII transport vesicles and in clustering COPII components at transitional ER (tER) sites. Sec16 interacts with multiple COPII components. Although the COPII assembly pathway is evolutionarily conserved, Sec16 homologues have not been described in higher eukaryotes. Here, we show that mammalian cells contain two distinct Sec16 homologues: a large protein that we term Sec16L and a smaller protein that we term Sec16S. These proteins localize to tER sites, and an N-terminal region of each protein is necessary and sufficient for tER localization. The Sec16L and Sec16S genes are both expressed in every tissue examined, and both proteins are required in HeLa cells for ER export and for normal tER organization. Sec16L resembles yeast Sec16 in having a C-terminal conserved domain that interacts with the COPII coat protein Sec23, but Sec16S lacks such a C-terminal conserved domain. Immunoprecipitation data indicate that Sec16L and Sec16S are each present at multiple copies in a heteromeric complex. We infer that mammalian cells have preserved and extended the function of Sec16.     

Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae

Golgi stacks are often located near sites of "transitional ER" (tER), where COPII transport vesicles are produced. This juxtaposition may indicate that Golgi cisternae form at tER sites. To explore this idea, we examined two budding yeasts: Pichia pastoris, which has coherent Golgi stacks, and Saccharomyces cerevisiae, which has a dispersed Golgi. tER structures in the two yeasts were visualized using fusions between green fluorescent protein and COPII coat proteins. We also determined the localization of Sec12p, an ER membrane protein that initiates the COPII vesicle assembly pathway. In P. pastoris, Golgi stacks are adjacent to discrete tER sites that contain COPII coat proteins as well as Sec12p. This arrangement of the tER-Golgi system is independent of microtubules. In S. cerevisiae, COPII vesicles appear to be present throughout the cytoplasm and Sec12p is distributed throughout the ER, indicating that COPII vesicles bud from the entire ER network. We propose that P. pastoris has discrete tER sites and therefore generates coherent Golgi stacks, whereas S. cerevisiae has a delocalized tER and therefore generates a dispersed Golgi. These findings open the way for a molecular genetic analysis of tER sites.     

Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif

Exit from the endoplasmic reticulum (ER) often occurs at distinct sites of vesicle formation known as transitional ER (tER) that are enriched for COPII vesicle coat proteins. We have characterized the organization of ER export in the malaria parasite, Plasmodium falciparum , by examining the localization of two components of the COPII machinery, PfSec12 and PfSec24a. PfSec12 was found throughout the ER, whereas the COPII cargo adaptor, PfSec24a, was concentrated at distinct foci that likely correspond to tER sites. These foci were closely apposed to cis -Golgi sites marked by PfGRASP–GFP, and upon treatment with brefeldin A they accumulated a model cargo protein via a process dependent on the presence of an intact diacidic export motif. Our data suggest that the cargo-binding function of PfSec24a is conserved and that accumulation of cargo in discrete tER sites depends upon positive sorting signals. Furthermore, the number and position of tER sites with respect to the cis -Golgi suggests a co-ordinated biogenesis of these domains.     

Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris

The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of ³²P-labeled sphingosine 1-phosphate.     

毕赤酵母过氧化物酶体吞噬的研究进展

过氧化物酶体吞噬是生物体的一种重要自我调控方式。过氧化物酶体吞噬是多种吞噬相关蛋白的共同作用,而且吞噬相关蛋白质之间的作用具有严格的时序性。由于毕赤酵母有两种吞噬方式、全基因组序列已知、基因操作技术成熟,所以毕赤酵母是研究过氧化物酶体吞噬的良好素材,也是目前研究的热点。本文对近年来毕赤酵母过氧化物酶体吞噬的启动、两种吞噬类型的形成、过氧化物酶体在液泡中降解的研究进行梳理,为毕赤酵母过氧化物酶体吞噬的进一步研究奠定基础。     

The crystal structure of Pichia pastoris lysyl oxidase

Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.     

毕赤酵母菌高效表达乙肝表面抗原的研究

目的:筛选高效表达HBsAg的毕赤酵母茵,制备目的蛋白.方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达栽体pPICZA中.构建重组质粒pPICZA-S和pPICZA-SH,经Sac I线性化后,LiCI化学法转化入酵母茵株GS115、X-33、KM71H和SMD1168.结果:诱导表达后的GS115工程茼单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72 h.结论:经SDS-PAGE和Westcrn-blot分析表明,所得产物为乙肝表面抗原S蛋白.     

The engagement of Sec61p in the ER dislocation process

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.     

人白介素12亚基p40和p35基因串联在毕赤酵母中的表达及表达产物活性分析

摘 要 利用毕赤酵母系统表达有活性的人单链白细胞介素12（hscIL-12）,PCR法从质粒pBI121-IL-12中扩增hscIL-12基因,经酶切、连接构建重组表达载体pPIC9K-hscIL-12,SacI线性化后,PEG1000法转化毕赤酵母GS115,经G418筛选和菌落PCR鉴定,经甲醇诱导,hscIL-12在酵母中获得分泌表达,表达产物经Western Blot检测,显示该蛋白相对分子质量为70KDa,可与鼠抗人IL-12单克隆抗体特异性结合;定量分析结果表明,重组酵母培养上清中hscIL-12约占总蛋白的26％,表达量约为60mg／L;生物学活性实验表明,重组蛋白能促进人外周血淋巴细胞增殖。为利用rhscIL-12进行基因治疗奠定了基础。     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B₂ receptor (B₂R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast Pichia pastoris. In the expression vector, B₂R gene was driven under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and Western blot analysis, it was proved that B₂R recombinant receptor proteins were expressed at a high level in the yeast. Furthermore, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B₂R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy. The text was submitted by the authors in English.     

毕赤酵母基因工程菌胞内AOX酶的检测方法

巴斯德毕赤酵母(Pichia pastoris)作为外源基因的表达宿主,已成功表达出一系列胞内和胞外蛋白[1～6],并已建立起了一套较成熟的发酵工艺.巴斯德毕赤酵母基因工程菌的外源基因,由胞内AOX酶(乙醇氧化酶)基因启动子调控.在非甲醇碳源条件下(如甘油或葡萄糖),AOX酶基因表达被抑制,外源基因也处于不表达状态.而以甲醇为唯一碳源时,AOX酶在胞内大量合成,同时外源基因被调控表达.在一般情况下,AOX酶的变化直接反映了外源基因的表达状况,因此通过分析检测胞内AOX酶的含量和变化速率,就可以确定外源基因所处的状态.     

毕赤酵母Gpn12异戊烯基转移酶NovQ催化合成MK-3

对异戊烯基转移酶NovQ在毕赤酵母Gpn12异源表达过程中诱导剂甲醇添加量进行了探究,并以毕赤酵母Gpn12全细胞为酶源,以甲萘醌、异戊烯醇为前体,催化合成维生素K2(MK-3)。每24 h添加2%甲醇时,NovQ表达量提高约36%。考察摇瓶中初始pH、温度、甲醇添加量、前体(甲萘醌、异戊烯醇)添加量、催化时间、十六烷基三甲基溴化铵(CTAB)添加量等7个因素对Gpn12全细胞催化合成MK-3的影响,发现催化温度、甲萘醌添加量、催化时间影响显著,对3个显著因素进行响应面优化得出催化条件为:催化温度31.56℃,甲萘醌添加量295.54 mg/L,催化时间15.87 h,优化后的摇瓶MK-3产量达到98.47 mg/L,与响应面预测结果一致,较优化前对照组提高了35%。在30 L发酵罐进行生物催化实验,催化时间24 h,细胞催化剂浓度220 g(干重)/L,MK-3产量达到189.67 mg/L。该方法为Gpn12规模化生产MK-3奠定了一定的基础。     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B2 receptor (B2R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast of Pichia pastoris. In the expression vector, B2R gene was drove under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and western blot analysis, it was proved that B2R recombinant receptor proteins were expressed at high level in the yeast. Further more, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B2R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy.     

Genotyping of recombinant Pichia pastoris strains

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.