Mullen R T, Trelease R N
Department of Plant Biology, Arizona State University, Tempe, Arizona 85287-1601, USA.
J Biol Chem. 2000 May 26;275(21):16337-44. doi: 10.1074/jbc.M001266200.
Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (N(cytosol)-C(matrix)) integral membrane protein that functions in the regeneration of NAD(+) in glyoxysomes of germinated oilseeds and protection of peroxisomes in other organisms from toxic H(2)O(2). Recently we showed that cottonseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was interpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxisomal ER (pER). Here we report on the molecular signals responsible for sorting peroxisomal APX. Deletions or site-specific substitutions of certain amino acid residues within the hydrophilic C-terminal-most eight-amino acid residues (includes a positively charged domain found in most peroxisomal integral membrane-destined proteins) abolished sorting of peroxisomal APX to peroxisomes via pER. However, the C-terminal tail was not sufficient for sorting chloramphenicol acetyltransferase to peroxisomes via pER, whereas the peptide plus most of the immediately adjacent 21-amino acid transmembrane domain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting sequences) plus the peroxisomal APX C-terminal tail also sorted chloramphenicol acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD appears to confer the proper context required for the conserved positively charged domain to function within peroxisomal APX as an overlapping pER sorting signal and a membrane peroxisome targeting signal type 2.
过氧化物酶体抗坏血酸过氧化物酶（APX）是一种羧基尾锚定的II型（N（胞质溶胶）-C（基质））整合膜蛋白，其功能是在萌发油籽的乙醛酸循环体中再生NAD⁺，并保护其他生物体中的过氧化物酶体免受有毒H₂O₂的侵害。最近我们发现，棉籽过氧化物酶体APX在翻译后通过一个新的网状/环状膜网络从胞质溶胶分选到过氧化物酶体，该网络被解释为内质网（ER）的一个亚结构域，称为过氧化物酶体ER（pER）。在此，我们报告负责分选过氧化物酶体APX的分子信号。亲水性最末端八个氨基酸残基（包括大多数定位于过氧化物酶体整合膜的蛋白质中发现的带正电荷结构域）内的某些氨基酸残基的缺失或位点特异性替换，消除了过氧化物酶体APX通过pER分选到过氧化物酶体的过程。然而，C末端尾巴不足以通过pER将氯霉素乙酰转移酶分选到过氧化物酶体，而过氧化物酶体APX的肽加上紧邻的21个氨基酸跨膜结构域（TMD）的大部分则足以进行分选。用过氧化物酶体APX的TMD替换为人工TMD（缺乏假定的分选序列）加上过氧化物酶体APX的C末端尾巴，也能通过pER将氯霉素乙酰转移酶分选到过氧化物酶体，这表明过氧化物酶体APX的TMD不具备必需的分选信息。相反，TMD似乎赋予了保守的带正电荷结构域在过氧化物酶体APX中作为重叠的pER分选信号和膜过氧化物酶体靶向信号2发挥作用所需的适当环境。
