Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast
Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast
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DOI：
10.1007/s00438-003-0938-8
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发表时间：
2004-01-01
影响因子：
3.1
通讯作者：
Niwa, O
中科院分区：
文献类型：
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作者：
Miki, F;Kurabayashi, A;Niwa, O
在分裂酵母的间期细胞中，纺锤体极体(SPB)被认为通过一种未知的跨越核膜的机制与染色体着丝粒相连。为了阐明这一机制，我们对与Kms1和SAD1相互作用的蛋白质进行了双杂交筛选，Kms1和SAD1是SPB的构成膜结合成分，相互作用。分别鉴定了7个和26个基因，它们的产物可能与Kms1和SAD1相互作用。除Dlc1(14 kDa动力蛋白轻链的同系物)外，所有Kms1相互作用元件也与SAD1相互作用。在已鉴定的基因中，有已知的基因rhp9(+)/crb2(+)、ut6(+)、ags1(+)/mok1(+)、gst3(+)、kms2(+)和sid4(+)。KMS2(+)和SID4(+)的产物定位于SPB。这些新基因的特征是构建中断突变和基因产物的定位。其中两个假设是芽期酵母UFE1(编码t-SNARE)和SFH1(染色质重塑复合体的基本成分)的同源物，这两个同源物对细胞的存活至关重要。另外两个基因只是条件必需的，与SAD1(+)在基因上相互作用。其中一个被命名为SIF1(+)(代表SAD1相互作用因子)，是高温下正常的隔膜形成所必需的。在该基因过表达的细胞中表现出类似于WEE的表型。另一个基因apm1(+)的产物与笼蛋白包裹的小泡中接头蛋白复合体的中链非常相似。Apm1似乎是SPB分离和纺锤体形成所必需的，当产量过高时，Apm1倾向于在SPB处积累。它在功能上与其同系物Apm2和Apm4不同。这项研究中发现的其他新基因包括一个核孔蛋白基因和编码新的膜结合蛋白的基因，这些基因在遗传上与SAD1有关。我们发现，新鉴定的基因中没有一个是着丝粒/端粒聚集所必需的。
In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9(+)/ crb2(+), cut6(+), ags1(+)/ mok1(+), gst3(+), kms2(+), and sid4(+). The products of kms2(+) and sid4(+) localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1(+). One of these was named sif1(+) (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1(+), is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.