評估酵母菌過氧化體基質蛋白SPS19之定位訊號PTS1的鄰近序列對於其蛋白運輸的影響

過氧化體的基質蛋白需和過氧化體的訊息序列PTS１結合後才能進入過氧化體的基質內進行作用。PTS1 位於C 端，由3個高度保留的氨基酸序列S/K/L或其他的變異型组成。PTS1序列除了C端的3個胺基酸之外，至少還需要上游的9個胺基酸才能形成PTS1:Pex5p複合體。酵母菌的SPS19可以轉譯成2,4-dienoyl-CoA reductase，脂肪酸雙鍵的裂解需要2,4-dienoyl-CoA reductase。當酵母菌的SPS19的基因刪除後，會使酵母菌無法進行出芽生殖和發生細胞溶解情形。而SPS19的C端具有SKL過氧化體的PTS1序列。為了探討SPS19蛋白的PTS1是否也需要12個胺基酸，我們使用PCR改造之前實驗室所具有的pRS424-Gal-GFP-PTS1(包含20個胺基酸)質體，構築出包含pRS424-Gal-GFP-PTS1-12a(包含12個胺基酸)及pRS424-Gal-GFP-PTS1-3a(包含3個胺基酸)的質體，及不含PTS1的pRS424-Gal-GFP質體。經DNA定序後使用ClustalW2做序列比對，來確定DNA序列的正確性，接著送入酵母菌中表現，觀察融合蛋白的GFP-PTS1是否會進入過氧化體中。從實驗中可發現來自pRS424-Gal-GFP-PTS1和pRS424-Gal-GFP-PTS1-12a的GFP-PTS1蛋白可完全進入過氧化體內，來自pRS424-Gal-GFP-PTS1-3a的GFP-PTS1進入過氧化體能力較差，而來自pRS424-Gal-GFP的GFP則只存於細胞質中。上述結果指出具有12個胺基酸以上的SPS19之PTS1能較有效的引導蛋白進入過氧化體。

關鍵字

釀酒酵母；過氧化體；綠色螢光；定位訊號

並列摘要

Peroxisomal matrix proteins are imported from cytoplasm into peroxisomes through peroxisomal targeting signal 1 (PTS1). Peroxisomal targeting signal (PTS) type 1 (PTS1) is a C-terminal uncleaved tripeptide (serine-lysine-leucine [SKL] or variants). Analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for signal recognition in the PTS1:Pex5p receptor complex. The yeast SPS19 gene encoding the peroxisomally targeted 2,4-dienoyl-CoA reductase ，fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase。The Saccharomyces cerevisiae SPS19 deleted was reduction in sporulation and sensitive to lytic.SPS19 reveled a carboxyl-terminal SKL peroxisome targeting signal. In this study , we constructed pRS424-Gal-GFP-PTS1-12a，pRS424-Gal-GFP-PTS1-3ａ, and pRS424-Gal-GFP, transformed these plasmid into wild-type W303.1a and W303.1a-Δpex19 by yeast transformation technique .The GFP-PTS1 encoding these strains were assayed peroxisome formation by the fluorescence microscopy technique.pRS424-Gal-GFP-PTS1-12a and pRS424-Gal-GFP-PTS1-3a DNA sequence use ClustalW2 analysis to perform comparative sequencing .We find the expected results for the pRS424-Gal-GFP-PTS1-20a and pRS424-Gal-GFP-PTS1-12a have green fluorescence in peroxisome, but the pRS424-Gal-GFP-PTS1-3a has green fluorescence less than the pRS424-Gal-GFP-PTS1-20a and pRS424-Gal-GFP-PTS1-12a in peroxisome, and pRS424-Gal-GFP have green fluorescence in cytosol. These results suggest that the C-terminal 12 amino acids of SPS19 is better sequence reguired for effective protein transport into peroxisome.

並列關鍵字

Saccharomyces cerevisiae ； peroxisome ； GFP ； PTS1

參考文獻

1. Braverman, N.E., M.D. D'Agostino, and G.E. Maclean, Peroxisome biogenesis disorders: Biological, clinical and pathophysiological perspectives. Dev Disabil Res Rev, 2013. 17(3): p. 187-96.

2. Gabaldon, T., Peroxisome diversity and evolution. Philos Trans R Soc Lond B Biol Sci, 2010. 365(1541): p. 765-73.

3. Wanders, R.J. and H.R. Waterham, Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem, 2006. 75: p. 295-332.

4. Dreyer, C., et al., Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors. Cell, 1992. 68(5): p. 879-87.

5. Saleem, R.A., J.J. Smith, and J.D. Aitchison, Proteomics of the peroxisome. Biochim Biophys Acta, 2006. 1763(12): p. 1541-51.

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評估酵母菌過氧化體基質蛋白SPS19之定位訊號PTS1的鄰近序列對於其蛋白運輸的影響

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