【作者】 朱静；

【导师】 高向东；

【作者基本信息】 武汉大学 ， 生物学·微生物学， 2020， 博士

【摘要】 酿酒酵母在出芽生殖过程中进行着极性生长,即胞内细胞骨架发生定向排布,形成运输物质的“轨道”,使得包裹着各种物质的囊泡被运输至生长位点,参与新细胞质膜和细胞壁等组分的形成。四十年多年来,人们已经在酿酒酵母中鉴定出来很多个调控极性生长的蛋白质分子,然而,仍然有很多调控分子,尤其是那些在极性生长中起着微调作用的分子,尚未被发现。本实验室在前期研究中筛选得到一个新的调控极性生长的蛋白质分子Tos7,本论文围绕Tos7对其细胞学功能和胞内定位进行了鉴定。酿酒酵母Cdc42是调控极性生长的中心因子,参与调控细胞骨架的组织和囊泡融合。本实验室前期研究发现过量表达Cdc42的负调控因子Rga1的功能区段Rga1-C538（a.a.538-1007）可以造成严重的细胞生长缺陷,该过表达菌株的生长缺陷可以被高拷贝RHO3、SEC9、SSO1、SSO2、SRO7、SEC1和BOI2基因所挽救,这些基因均参与调控极性分泌,显示出该过表达菌株可能存在分泌缺陷。我们发现一个功能未知的基因TOS7（YOL019W）在高拷贝表达时,也能够高效地挽救过量表达Rga1-C538造成的细胞生长缺陷,本研究还发现高拷贝TOS7还能够挽救分泌功能缺陷的温度敏感型突变体sec15-1在限制性温度下的生长缺陷,显示Tos7是一个新的极性分泌调控因子。Tos7是一个跨膜蛋白,含有1个信号肽和3个跨膜区,属于Sur7/Pal I家族成员。该家族成员的氨基酸序列中同源性最高的区段是位于N端的信号肽和3个跨膜区,该区段被称为Sur7结构域。酿酒酵母含有10个Sur7/Pal I家族成员,它们的功能多种多样。Tos7的同源分子在酵母中广泛存在,但绝大多数同源分子的功能尚不清楚。令人感兴趣的是,Tos7在粟酒裂殖酵母中的同源分子Mug33被报道能够调控极性分泌,显示Tos7调控极性分泌的功能在进化中可能是保守的。序列比对分析显示Tos7与同家族成员中的Dcv1和Rim9最相似,三者在信号肽和第一个跨膜区之间均含有保守的Gly-Cys box。然而,酿酒酵母的10个Sur7/Pal I家族成员中,只有Tos7能够挽救过量表达Rga-C538造成的生长缺陷,Tos7的C端胞质区段也是这10个同源分子中最长的,长度达367个氨基酸。通过对Tos7进行截短,我们发现Tos7的N端Sur7结构域和C端胞质区段的前半段及邻近的含有Pxxx P富含脯氨酸的proline-rich（PR1）结构域对其调控分泌功能的发挥是必需的。当Sur7结构域中Gly-Cys box中两个保守的甘氨酸Gly 47或Gly 50发生突变(Tos7^G47D或Tos7^G50D)造成Tos7细胞质膜定位消失后,Tos7挽救过量表达Rga-C538细胞生长缺陷的能力也随之消失,暗示Sur7结构域介导的正确定位对于Tos7的功能发挥至关重要。我们还发现当PR1缺失后,Tos7的功能也急剧下降,推测Tos7可能通过胞质区段中PR1结构域与其它极性分泌调控因子相互作用,进而调控极性分泌。我们对Tos7调控极性分泌机制进行了探究,发现Tos7与Rho3和Boi2之间存在体内相互作用。Tos7和Rho3的激活型(Rho3^Q74L模拟)和失活型(Rho3^T30N模拟)均存在相互作用,没有偏好性,显示Tos7不是Rho3的下游效应因子。Tos7通过PR1结构域与Boi2中的SH3结构域发生相互作用。Rho3和Boi2被报道参与胞吐复合体的组装过程,Tos7在裂殖酵母中的同源分子Mug33也被报道可能是通过调控胞吐复合体组装参与极性分泌过程,鉴于此,我们推测Tos7可能通过其与Rho3和Boi2相互作用,从而参与胞吐复合体的组装,调控极性分泌的。我们发现缺失或过量表达TOS7均不会使细胞出现明显的生长缺陷,显示其对于细胞的生长不是必需的,推测Tos7可能对于极性生长具有微调的功能。Tos7定位在细胞质膜和液泡中。Tos7在细胞质膜上呈斑状分布,且与膜上亚结构域上MCC（Membrane Compartment of Can1）组分Sur7在极大程度上存在共定位,显示Tos7定位于细胞质膜上MCC处。我们发现Tos7在细胞质膜上定位主要依赖Sur7结构域。与构巢曲霉Pal I中Gly-Cys box中保守甘氨酸类似,我们发现Tos7的Gly-Cys box中的两个甘氨酸中任何一个发生突变,都会造成Tos7的质膜定位完全消失,显示Gly-Cys box对于Tos7的膜定位非常重要。Tos7的液泡定位则需要胞质区段,推测Tos7的胞质区段可能介导了其被内吞,但通过对潜在内吞化识别位点的氨基酸及区段进行突变,我们并未发现关键位点或区段,推测Tos7内吞可能是有多个氨基酸或区段共同介导。除了调控极性分泌,我们发现Tos7还影响细胞壁完整性。Tos7的Sur7结构域在Tos7影响细胞壁完整性方面起着主要作用,显示此功能与Tos7在极性分泌方面的功能是不同的。Tos7的Gly-Cys box中两个保守甘氨酸Gly 47或Gly 50发生突变后,Tos7影响细胞壁完整性的功能也完全丧失,显示Tos7的细胞质膜定位对其功能发挥至关重要。我们发现酿酒酵母Sur7/Pal I家族成员中与Tos7一样位于MCC处的分子如Sur7、Pun1和Fmp45均具有影响细胞壁完整性的功能,且Sur7与Tos7存在功能冗余。Tos7还与Sur7、Pun1和Fmp45在体内存在相互作用。考虑到一些与MCC稳定性相关的eisosome复合体中的某些成员也具有影响细胞壁完整性的功能,我们推测Tos7可能与MCC/eisosome的结构稳定性有关。我们还发现与Pun1一样,Tos7在体内存在自身相互作用,且此相互作用由Sur7结构域介导,我们推测Tos7的自身相互作用可能有助于MCC/eisosome的稳定。我们的研究结果揭示了Tos7的细胞学功能与胞内定位,第一次揭示了Sur7/Pal I家族中的一个成员能够调控极性分泌,为此家族其它成员的研究及其它物种中Tos7同源分子的研究提供了方向。更多还原

【Abstract】 The budding yeast Saccharomyces cerevisiae undergoes highly polarized growth during budding,in which the actin cytoskeleton is polarized and serves as the track for the delivery of secretory vesicles packed with cargos to the growing site.The newly delivered materials participate in the synthesis of new plasma membrane and the cell wall.For more than fourty years,researchers have identified many proteins that regulate polarized growth,yet many more,particularly those that play a fine-tuning role in polarized growth,remain elusive.Our research group has previously identified a novel regulator of polarized growth,Tos7,whose function was unknown.This thesis will focus on Tos7 to characterize its cellular functions and subcellular localization.Cdc42 is the central regulator of polarized growth.It regulates the assembly and polarization of the actin cytoskeleton and the fusion of secretory vesicles with the plasma membrane.Previously,our lab discovered that overexpression of the Rga1-C538（a.a.538-1007）segment of Rga1,a negative regulator of Cdc42,severely impaired cell growth.This growth defect can be rescued by high copy RHO3,SEC9,SSO1,SSO2,SRO7,SEC1 or BOI2,all of which regulate polarized secretion.This finding suggests that overexpression of Rga1-C538 may cause a defect in secretion in the cells.We found that high-copy expression of TOS7（YOL019W）,a gene with unknown function,also suppressed the growth defect of Rga1-C538-overexpressing cells.In addition,high-copy TOS7 also suppressed the growth defect of secretion-defective temperature-sensitive sec15-1 mutant at the restrictive temperature.These findings suggest that Tos7 is a novel regulator of polarized secretion.Tos7 is a transmembrane protein featuring a signal peptide and three transmembrane domains.Tos7 belongs to the Sur7/Pal I family.The region that shares the highest amino acid sequence similarity among the members of the Sur7/Pal I family is the Sur7 domain,which comprises the signal peptide and the three transmembrane domains.There are ten Sur7/Pal I family members that play diverse functions in the budding yeast Saccharomyces cerevisiae.Tos7 homologs are widespread in yeast.However,their functions are not well understood.Interestingly,Mug33,the Tos7 homolog in the fission yeast Schizosaccharomyces pombe,was reported to regulate polarized secretion,suggesting that the function of Tos7 in secretion might be evolutionarily conserved.Sequence alignment reveals that Tos7 is most similar to Dcv1 and Rim9.All of the three proteins contain a conserved Gly-Cys box between the signal peptide and the first transmembrane domain.However,among the ten Sur7/Pal I members in S.cerevisiae,only Tos7 suppressed the growth defecct of Rga1-C538-overexpressing cells when expressed in high copies.Tos7 has the longest,367-a.a.long cytoplasmic tail among the ten Sur7/Pal I family members.By using trunctated forms of Tos7,we found that the N-terminally located Sur7 domain and the first half of the cytoplasmic tail as well as a neighboring Pxxx P motif-containing proline-rich PR1 domain are critical for Tos7’s function in the regulation of secretion.When the two conserved glycine residues in the Gly-Cys box,Gly 47 or Gly50,was mutated to Asp,which causes a disruption of the plasma-membrane localizationof Tos7,Tos7no longer suppressed the growth defect of Rga1-C538-overexpressing cells.This result suggests that the proper localization of Tos7 on the plasma membrane is essential for Tos7’s function.We found that the deletion of the proline-rich PR1 domain also impaired Tos7’s function.We speculate that Tos7 may regulate polarized secretion via the interaction of PR1with regulators of secretion.We have investigated the mechanism by which Tos7 regulates polarized secretion.We found that Tos7 physically interacts with Rho3 and Boi2.Tos7 interacts with both the active GTP-bound form(mimmicked by Rho3^Q74L)and the inactive GDP-bound form(mimmicked by Rho3^T30N)of Rho3 without a preference for either of them,indicating that Tos7 is not a downstream effector of Rho3.Tos7 interacts with Boi2 via the PR1 domain of Tos7 and the SH3 domain of Boi2.Rho3 and Boi2 function in the assembly of the exocyst complex.Mug33,the Tos7 homolog in the fission yeast,is also thought to regulate polarized secretion by the regulation of exocyst assembly.Based on these findings,we speculate that Tos7 may regulate polarized secretion via its interaction with Rho3 and Boi2,which are involved in the assembly of the exocyst complex.We found that the neither the deletion of TOS7 nor TOS7overexpression impaired cell growth,suggesting that Tos7 is not essential for cell growth.Tos7may play a fine-tuning role in polarized growth.Tos7 localizes to the plasma membrane and the vacuoles.Tos7 localizes to patches that largely co-localize with the MCC（Membrane Compartment of Can1）component Sur7,indicating that Tos7 localizes to MCC.We found that the plasma membrane localization of Tos7 is mainly mediated by the Sur7 domain.Similar to the conserved glycine residues in the Gly-Cys box of Pal I in Aspergillus nidulans,mutation of either of the two glycine residues in the Gly-Cys box of Tos7 completely eliminated Tos7’s plasma membrane localization,suggesting that the Gly-Cys box is required for Tos7’s plasma membrane localization.Tos7’s vacuolar localization requires the cytoplasmic tail.We speculate that the cytoplasmic tail of Tos7 may mediate the internalization of Tos7 into the cytoplasm.Unfortunately,by mutagenesis of putative amino acid residues,we failed to identify the key amino acid residues or regions within Tos7 that are important for Tos7 internalization.We speculate that the internalization of Tos7 may be mediated by multipe amino acid reidues or regions.In addition to secretion,we found that Tos7 also affects cell wall integrity.The Sur7domain plays an essential role for Tos7 in cell wall integrity,which is in sharp contrast to Tos7’s role in polarized secretion.Mutation of either of the two conserved glycine residues,Gly 47 or Gly 50,within the Gly-Cys box of Tos7 completely abolished Tos7’s function in cell wall integrity,indicating that the plasma membrane localization of Tos7 is essential for Tos7 in cell wall integrity.We found that the Sur7/Pal I family members in S.cerevisiae that localize to MCC like Tos7 does such as Sur7,Pun1 and Fmp45 also affect cell wall integrity.Sur7 is functionally redundant with Tos7.Tos7 also physically interacts with Sur7,Pun1 and Fmp45in vivo.Considering that some MCC/eisosome components also affect cell wall integrity,we speculate that Tos7 might be involved in the structural integrity of MCC/eisosome.Like Pun1,Tos7 also interacts with itself in vivo.This homotypic interaction of Tos7 is mediated by the Sur7 domain.We speculate that the homotypic interaction of Tos7 may help to stabilize the structure of MCC/eisosome.Our results reveal the cellular functions and the subcellular localization of Tos7.This is the first report that a Sur7/Pal I family member regulates secretion in S.cerevisiae.These results will shed light on the understanding of the cellular functions of Tos7 orthologs in different yeast species.更多还原