Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY-2 glyoxysomes
Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY-2 glyoxysomes
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DOI：
10.1046/j.1365-313x.1998.00344.x
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发表时间：
1998-12-01
期刊：
影响因子：
7.2
通讯作者：
Trelease, RN
中科院分区：
文献类型：
--
作者：
Flynn, CR;Mullen, RT;Trelease, RN
在这项研究中，我们检测了pts2-九肽中残基的明显不连续性和保守性，并证明了这种拓扑信号能够指导异源多聚体蛋白在植物细胞中的输入。根据累积的数据显示，在12种不同的过氧化体目标蛋白中至少存在26个独特的推测的PTS2非肽，当前的(-R/K-L/V/I-X-5-H/Q-L/A-)以及原始的(-R-L-X-5-H/Q-L-)PTS2基序似乎被过度简化了。为了评估基序中残基的功能，在悬浮培养烟草中瞬时表达了大鼠肝脏硫解酶(Rthio)和各种嵌合的氯霉素乙酰转移酶(CAT)蛋白。CV Bright Huang Cells(BY-2)，免疫荧光显微镜观察其亚细胞定位。缺乏pts2的血凝素(HA)表位标记的CAT亚基(CAT-HA)被携带pts2的CAT亚基(rthio-CAT)‘携带’到乙氧体中，而信号耗竭的CAT-HA亚基被修饰以防止寡聚化而不进入乙氧体，这些结果提供了直接的证据，证明信号耗竭的亚基被pts2作为寡聚体(异构体)靶向细胞器。对pts2非编码肽中残基的突变分析表明，许多氨基酸替换能够维持靶向功能。此外，pts2九肽内残基的功能似乎不需要相邻残基赋予的上下文特定环境。这些结果综合表明，功能PTS2不仅定义为序列特异性基序，即-R/K-X-6-H/Q-A/L/F-，还定义了其结构基序，该结构基序取决于九肽中残基的物理化学性质。
In this study of the type 2 peroxisomal targeting signal (PTS2) pathway, we examined the apparent discontinuity and conservation of residues within the PTS2 nonapeptide and demonstrated that this topogenic signal is capable of directing heteromultimeric protein import in plant cells. Based on cumulative data showing that at least 26 unique, putative PTS2 nonapeptides occur within 12 diverse peroxisomal-destined proteins, the current (-R/K-L/V/I-X-5-H/Q-L/A-) as well as the original (-R-L-X-5-H/Q-L-) PTS2 motif appear to be oversimplified. To assess the functionality of residues within the motif, rat liver thiolase (rthio) and various chimeric chloramphenicol acetyltransferase (CAT) proteins were expressed transiently in suspension-cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow cells (BY-2), and their subcellular location was determined by immunofluorescence microscopy. Hemagglutinin (HA)epitope-tagged-CAT subunits, lacking a PTS2 (CAT-HA), were 'piggybacked' into glyoxysomes by PTS2-bearing CAT subunits (rthio-CAT), whereas signal-depleted CAT-HA subunits that were modified to prevent oligomerization did not import into glyoxysomes, These results provided direct evidence that signal-depleted subunits imported into peroxisomes were targeted to the organelle as oligomers (heteromers) by a PTS2. Mutational analysis of residues within PTS2 nonapeptides revealed that a number of amino acid substitutions were capable of maintaining targeting function. Furthermore, functionality of residues within the PTS2 nonapeptide did not appear to require a context-specific environment conferred by adjacent residues. These results collectively suggest that the functional PTS2 is not solely defined as a sequence-specific motif, i.e. -R/K-X-6-H/Q-A/L/F-, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.