Wolins N E, Donaldson R P
Department of Biological Sciences, George Washington University, Washington, DC 20052, USA.
Plant Physiol. 1997 Mar;113(3):943-9. doi: 10.1104/pp.113.3.943.
The carboxyl-terminal amino acid sequence serine-lysine-leucine (SKL) is the consensus peroxisomal targeting sequence 1 (PTS1) and is sufficient to direct a polypeptide to peroxisomes in vivo in plants, animals, and yeasts. However, there are also two sites on alkali-stripped glyoxysomal membranes from castor bean (Ricinus communis) endosperm that bind the peptide YHKHLKPLQSKL (SKLp), the sequence of the last 12 amino acids of acyl-coenzyme A oxidase (N.E. Wollins, R.P. Donaldson [1994] J Biol Chem 289: 1149-1153). It was hypothesized that one of these sites interacts with information other than the PTS1. To explore the sequence requirements for each SKLp binding site, we tested the peptides YHKHLKPQSKG and YHKHLKPLQS and found that they bound to the high-affinity site, but not to the low-affinity site. When the high-affinity site was blocked with YHKHLKPQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 microM. In an attempt to disrupt high-affinity binding, two the upstream, positively charged residues were replaced with negatively charged residues to make the peptide YHKETEPLQSKL. YHKETEPLQSKL did not bind to either site on the glyoxysomal membranes. These results indicate that the PTS1 binds to the low-affinity site and that the adjacent, positively charged domain binds to the high-affinity site.
羧基末端氨基酸序列丝氨酸 - 赖氨酸 - 亮氨酸（SKL）是公认的过氧化物酶体靶向序列1（PTS1），足以在植物、动物和酵母体内将多肽导向过氧化物酶体。然而，蓖麻（Ricinus communis）胚乳经碱处理的乙醛酸循环体膜上也有两个位点可结合肽YHKHLKPLQSKL（SKLp），即酰基辅酶A氧化酶最后12个氨基酸的序列（N.E. Wollins，R.P. Donaldson [1994] J Biol Chem 289: 1149 - 1153）。据推测，其中一个位点与PTS1以外的信息相互作用。为了探究每个SKLp结合位点的序列要求，我们测试了肽YHKHLKPQSKG和YHKHLKPLQS，发现它们能结合高亲和力位点，但不能结合低亲和力位点。当高亲和力位点被YHKHLKPQSKG阻断时，SKLp以8.5微摩尔的解离常数（Kd）结合到低亲和力位点。为了破坏高亲和力结合，将两个上游带正电荷的残基替换为带负电荷的残基，得到肽YHKETEPLQSKL。YHKETEPLQSKL不能结合乙醛酸循环体膜上的任何一个位点。这些结果表明，PTS1结合低亲和力位点，而相邻的带正电荷结构域结合高亲和力位点。
