Hudak K A, Dinman J D, Tumer N E
Biotechnology Center for Agriculture and the Environment and Department of Plant Pathology, Rutgers University, New Brunswick, New Jersey 08901-8520, USA.
J Biol Chem. 1999 Feb 5;274(6):3859-64. doi: 10.1074/jbc.274.6.3859.
Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein, catalytically removes an adenine residue from the conserved alpha-sarcin loop of the large rRNA, thereby preventing the binding of eEF-2.GTP complex during protein elongation. Because the alpha-sarcin loop has been placed near the peptidyltransferase center in Escherichia coli ribosomes, we investigated the effects of alterations at the peptidyltransferase center on the activity of PAP. We demonstrate here that a chromosomal mutant of yeast, harboring the mak8-1 allele of peptidyltransferase-linked ribosomal protein L3 (RPL3), is resistant to the cytostatic effects of PAP. Unlike wild-type yeast, ribosomes from mak8-1 cells are not depurinated when PAP expression is induced in vivo, indicating that wild-type L3 is required for ribosome depurination. Co-immunoprecipitation studies show that PAP binds directly to L3 or Mak8-1p in vitro but does not physically interact with ribosome-associated Mak8-1p. L3 is required for PAP to bind to ribosomes and depurinate the 25 S rRNA, suggesting that it is located in close proximity to the alpha-sarcin loop. These results demonstrate for the first time that a ribosomal protein provides a receptor site for an ribosome-inactivating protein and allows depurination of the target adenine.
商陆抗病毒蛋白（PAP）是一种29 kDa的核糖体失活蛋白，它能催化去除大核糖体RNA保守的α-肌动蛋白环中的一个腺嘌呤残基，从而在蛋白质延伸过程中阻止eEF-2·GTP复合物的结合。由于α-肌动蛋白环已定位在大肠杆菌核糖体的肽基转移酶中心附近，我们研究了肽基转移酶中心的改变对PAP活性的影响。我们在此证明，携带肽基转移酶连接的核糖体蛋白L3（RPL3）的mak8-1等位基因的酵母染色体突变体对PAP的细胞生长抑制作用具有抗性。与野生型酵母不同，当在体内诱导PAP表达时，来自mak8-1细胞的核糖体不会发生脱嘌呤作用，这表明野生型L3是核糖体脱嘌呤所必需的。免疫共沉淀研究表明，PAP在体外直接与L3或Mak8-1p结合，但不与核糖体相关的Mak8-1p发生物理相互作用。L3是PAP结合核糖体并使25 S rRNA脱嘌呤所必需的，这表明它位于靠近α-肌动蛋白环的位置。这些结果首次证明，一种核糖体蛋白为核糖体失活蛋白提供了一个受体位点，并允许靶腺嘌呤的脱嘌呤作用。
