Chen L, Johnson R C, Milgram S L
Department of Cell and Molecular Physiology, The University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 1998 Dec 11;273(50):33524-32. doi: 10.1074/jbc.273.50.33524.
The cytosolic domain of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) contains signals that direct its trafficking in the secretory and endosomal pathways. Using the yeast two-hybrid system, Alam et al. (Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636) identified three proteins that interact with a fragment of the PAM cytosolic domain containing these targeting signals. We cloned the rat and human cDNAs encoding PAM COOH-terminal interactor protein-1 (P-CIP1). Both cDNAs contain an open reading frame that encodes a novel protein of 435 amino acids. The P-CIP1 protein is highly conserved from rat to human (85% identity) but does not display significant homology to proteins in the GenBank data base. In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1, but does not interact with mutant PAM-1 proteins that fail to target correctly when expressed in endocrine cells. P-CIP1 contains multiple consensus serine/threonine phosphorylation sites and a region predicted to form a coiled-coil at the COOH terminus. When expressed in endocrine cells or fibroblasts, P-CIP1 is distributed in a punctate pattern in the perinuclear area but does not significantly overlap the distribution of transfected wild type PAM-1. The distribution of P-CIP1 displays significant overlap with the distribution of the secretory carrier membrane proteins, internalized Texas Red-conjugated transferrin, and Rab11. The data suggest that P-CIP1 associates with vesicles in the recycling endosomal pathway, and may play a role in regulating the trafficking of integral membrane PAM.
肽加工酶肽基甘氨酸α-酰胺化单加氧酶（PAM）的胞质结构域含有指导其在分泌和内体途径中运输的信号。Alam等人（Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636）利用酵母双杂交系统鉴定出三种与包含这些靶向信号的PAM胞质结构域片段相互作用的蛋白质。我们克隆了编码PAM羧基末端相互作用蛋白-1（P-CIP1）的大鼠和人类cDNA。两种cDNA均包含一个开放阅读框，该开放阅读框编码一种由435个氨基酸组成的新型蛋白质。P-CIP1蛋白在大鼠和人类之间高度保守（同一性为85%），但与GenBank数据库中的蛋白质没有显著同源性。在体外，P-CIP1与野生型PAM-1的胞质结构域相互作用，但不与在内分泌细胞中表达时无法正确靶向的突变型PAM-1蛋白相互作用。P-CIP1包含多个共有丝氨酸/苏氨酸磷酸化位点以及一个预计在羧基末端形成卷曲螺旋的区域。当在内分泌细胞或成纤维细胞中表达时，P-CIP1以点状模式分布于核周区域，但与转染的野生型PAM-1的分布没有明显重叠。P-CIP1的分布与分泌载体膜蛋白、内化的德克萨斯红偶联转铁蛋白和Rab11的分布有显著重叠。数据表明，P-CIP1与再循环内体途径中的囊泡相关联，可能在调节整合膜PAM的运输中发挥作用。
