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。

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-PTS1-12ahave green fluorescence in peroxisome, but thepRS424-Gal-GFP-PTS1-3a has green fluorescence less than the pRS424-Gal-GFP-PTS1-20a and pRS424-Gal-GFP-PTS1-12ain 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.