The carboxyl-terminal region of protein C is essential for its secretion.
We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.
Katsumi
A
A
First Department of Internal Medicine and the First Department of Anatomy, Nagoya University School of Medicine, Nagoya, Japan. akira@darwin.wustl.edu
Kojima
T
T
Senda
T
T
Yamazaki
T
T
Tsukamoto
H
H
Sugiura
I
I
Kobayashi
S
S
Miyata
T
T
Umeyama
H
H
Saito
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D046911:Macromolecular Substances; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011486:Protein C; C112398:protein C Nagoya, human