Flynn C R, Mullen R T, Trelease R N
Graduate Program in Molecular and Cellular Biology, Arizona State University, Tempe 85287-1601, USA.
Plant J. 1998 Dec;16(6):709-20. doi: 10.1046/j.1365-313x.1998.00344.x.
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-X5-H/Q-L/A-) as well as the original (-R-L-X5-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 tabaccum L.) cv Bright Yellow cells (BY-2), and their subcellular location was determined by immunofluoresence 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-X6-H/Q-A/L/F-, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.
在这项关于2型过氧化物酶体靶向信号（PTS2）途径的研究中，我们检测了PTS2九肽内残基的明显间断性和保守性，并证明这种拓扑信号能够在植物细胞中引导异源多聚体蛋白的导入。基于累积数据显示，在12种不同的定位于过氧化物酶体的蛋白中至少出现26种独特的、推定的PTS2九肽，当前的（-R/K-L/V/I-X5-H/Q-L/A-）以及最初的（-R-L-X5-H/Q-L-）PTS2基序似乎都过于简化了。为了评估基序内残基的功能，将大鼠肝脏硫解酶（rthio）和各种嵌合氯霉素乙酰转移酶（CAT）蛋白瞬时表达于悬浮培养的烟草（Nicotiana tabaccum L.）品种Bright Yellow细胞（BY-2）中，并通过免疫荧光显微镜确定它们的亚细胞定位。缺乏PTS2的血凝素（HA）表位标记的CAT亚基（CAT-HA），通过携带PTS2的CAT亚基（rthio-CAT）“搭载”进入乙醛酸循环体，而经过修饰以防止寡聚化的信号缺失的CAT-HA亚基则不会导入乙醛酸循环体。这些结果提供了直接证据，表明导入过氧化物酶体的信号缺失亚基作为寡聚体（异源体）通过PTS2靶向到该细胞器。对PTS2九肽内残基的突变分析表明，许多氨基酸取代能够维持靶向功能。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异性环境。这些结果共同表明，功能性PTS2不仅仅被定义为序列特异性基序，即-R/K-X6-H/Q-A/L/F-，还由其结构基序定义，该结构基序取决于九肽内残基的物理化学性质。
