Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris.
Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris.
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DOI：
10.1083/jcb.139.6.1419
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发表时间：
1997-12-15
期刊：
影响因子：
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通讯作者：
Cregg JM
中科院分区：
文献类型：
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作者：
Waterham HR;Russell KA;Vries Y;Cregg JM
醇氧化酶（AOX）是酵母甲醇利用途径中的第一个酶，是一种同型八聚体过氧化物酶体基质蛋白。在酵母毕赤酵母的过氧化物酶体生物合成缺陷（pex）突变体中，AOX不能组装成活性八聚体，而是形成无活性的细胞质聚集体。AOX明显不能在细胞质中组装，这与其他过氧化物酶体蛋白在输入前能够寡聚化形成对比。为了进一步研究AOX的作用，我们首先鉴定了它的过氧化物酶体靶向信号（PTS）。我们发现靶向AOX所必需的序列主要位于亮氨酸-丙氨酸-丙氨酸-苯丙氨酸COOH（LARF）蛋白质的4个COOH末端氨基酸内。为了检测AOX是否可以在导入前发生寡聚化，我们共表达了AOX（没有PTS）和野生型AOX（沿着），并确定了突变型AOX是否可以共导入过氧化物酶体。为了鉴定AOX的突变形式，用血凝素表位标签（AOX-HA）替换蛋白质的COOH末端LARF序列。AOX-HA与野生型AOX（AOX-WT）的共表达没有导致八聚体活性AOX中存在的AOX-HA的比例增加，表明新合成的AOX-HA不能与AOX-WT在细胞质中寡聚化。因此，AOX不能在细胞质中启动寡聚化，但必须首先在组装开始之前靶向细胞器。
Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.