酵母菌Hsp26蛋白在原生孢子膜形成時的作用

減數分裂在有性生殖中扮演重要的角色。在營養缺乏的環境中，二倍體的酵母菌 (Saccharomyces cerevisiae) 細胞會經由減數分裂產生四個單倍體的孢子。整個減數分裂和孢子形成 (spore formation) 的過程稱為孢子生殖 (sporulation)。在孢子生殖過程中熱休克蛋白 Hsp26 會大量的表現，過去研究發現 hsp26 突變株的產孢率比野生型高，由於減數分裂沒有明顯的差異，表示缺少 Hsp26 是影響孢子形成時期。此外 hsp26 突變株中不正常排列的孢子比例較多，孢子的排列受到紡錘體 (spindle) 的控制，因此使用 benomyl 擾亂野生型與 hsp26 突變株的紡錘體，發現 hsp26 突變株的產孢率沒有受到影響，而野生型則有明顯的降低。根據此結果，之前推測 Hsp26 可能參與在紡錘體相關的檢控點。本論文的主要目的在於更深入探討 Hsp26 調控孢子形成的機制。首先，我們研究可能和 Hsp26 交互作用而且參與在孢子形成時期的蛋白質：Ady3。Ady3 位在原生孢子膜 (prospore membrane; PSM) 的前緣蛋白鞘 (leading edge protein coat; LEP coat) 上，與原生孢子膜的形成及孢子壁合成有關。根據之前的研究發現在 hsp26 突變株中偵測到較多 Ady3 位在前緣蛋白鞘上，因此推測 Hsp26 可能藉由調控Ady3結合到前緣蛋白鞘上，進而控制了產孢過程。此外，過去的研究顯示 Ady3 靠著 Ssp1 結合到前緣蛋白鞘上，故推測出第二種假設，Hsp26 可能經由調控 Ssp1而影響到 Ady3與前緣蛋白鞘的結合。以 benomyl 或低溫擾亂紡錘體後，經由免疫螢光觀察，發現野生型中 Ssp1 結合到前緣蛋白鞘上的比例均會降低，但是在 hsp26 突變株中則未降低，結果與第二種假設相符。我們推測在酵母菌的產孢過程中，當紡錘體異常時 Hsp26 可能會受到紡錘體相關檢控點的訊號，進而參與在調控 Ssp1 結合到前緣蛋白鞘上的過程。

關鍵字

Hsp26 蛋白；原生孢子膜；前緣蛋白鞘；紡垂體檢控點；減數分裂

並列摘要

Meiosis plays an important role in gametogenesis. A diploid budding yeast cell enters meiosis under nutrient starvation which finally produces four haploid spores. The overall process of meiosis and spore formation is called sporulation. During this process, heat shock protein Hsp26 was strongly induced. Previous studies found that the sporulation efficiency of hsp26 mutant is higher than that of the wild type, and this increase occurs at the step of spore formation. Also, there is an increase in the percentages of abnormal asci in hsp26 mutant cells; asci alignment depends on the meiotic spindle functions. In order to specificially disrupt meiotic spindles, low level of benomyl was used. It caused a reduction of spore formation in the wild type, but not in the hsp26 mutant cells. Thus it was proposed that Hsp26 might be involved in the spindle checkpoint controlling the transition from meiosis II to spore formation. To understand the molecular mechanism of how Hsp26 might be involved in the spindle checkpoint during spore formation, we studied a potential Hsp26-interacting protein, Ady3. Ady3 is a component of the leading edge protein (LEP) coat of the prospore membrane (PSM) and is important for PSM formation and spore wall synthesis. Previous studies found that more Ady3 signals were detected on the LEP coat in the hsp26 mutant. Taken together, we hypothesized that Hsp26 might be involved in the spindle checkpoint by regulating Ady3 localization onto the leading edge coat, thus preventing the formation of abnormal asci. In addition, localization dependence study showed that Ady3 binding to the leading edge depends on Ssp1. Therefore, we also hypothesized the other possibilty that Ssp1 might act as the regulation target of Hsp26 in controlling spore formation. Upon microtubule-perturbation treatments, we found that the localization of Ssp1 to the LEP coat is affected in wild type cells, but not in hsp26 mutant cells. Based on these results, we proposed that in response to the spindle checkpoint signaling, Hsp26 might be involved in the regulation of Ssp1 for controlling spore formation.

並列關鍵字

Hsp26 ； prospore membrane ； leading-edge complex ； spindle checkpoint ； meiosis

參考文獻

Arrigo, A.P. and Landry, J. (1994). Expression and function of the low molecular weight heat shock proteins. In Morimoto, R.I. (ed.), The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 335-373.

Google Scholar

Boeke, J.D., Trueheart, J., Natsoulis, G. and Fink, G.R. (1987). 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154: 164-175.