A signal for retention of cytochrome P450 2C1 in the endoplasmic reticulum

To determine whether the N-terminal hydrophobic sequence is also responsible for retention, the N-terminal 29 amino acids of cytochrome P450 2C1, with and without an additional 29 amino acids containing an N-glycosylation site, were fused to a soluble cytoplasmic protein, Escherichia coli β-galactosidase, or to a secreted protein, Escherichia coli alkaline phosphatase, and the hybrid proteins were expressed in COS1 cells and cellular localization was determined by subcellular fractionation, immunolocalization and the glycosylation state of the proteins. Both the β-galactosidase and alkaline phosphatase hybrid proteins were retained in the endoplasmic reticulum establishing that a specific sequence or property in the N-terminal 29 amino acids is responsible for ER retention. To further examine the possibility that retention of proteins with a large cytoplasmic domain is the default pathway, the cellular distributions of a series of P450 2C1 and EGFR chimeric proteins were examined. The data indicate that the cytoplasmic domain of the protein also has retention properties. Protein containing only the transmembrane domain of EGFR at the amino-terminus and the EGFR cytoplasmic domain were transported to the plasma membrane, while the equivalent construction with the cytoplasmic domain of cytochrome P450 was retained in the ER. The reciprocal construction with the N-terminal sequence of P450 and the cytoplasmic domain of EGFR was retained in the ER confirming the previous studies that the N-terminal sequence has ER retention properties. The retention properties of the normally N-terminal 29 amino acids was dependent on their position in the protein. We subjected the N-terminal region of P450 to extensive mutagenesis to further define the specific sequence requirements for retention. We demonstrated that the C-terminal flanking sequences following the transmembrane domain are not important for ER retention. Substitutions of either leucine or alanine for non-leucine residues did not alter the ER retention properties of the transmembrane domain. Therefore, it seems likely that another property like secondary structure, hydrophobicity, hydrophilicity, or length of the transmembrane domain of P450 may be important for ER retention.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular and Integrative Physiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Ahn, Kwangseog

Contributors dc:contributor

• Kemper, Byron W.

• Biology, Cell
• Biology, Animal Physiology

Statement dc:rights

• Copyright 1994 Ahn, Kwangseog

Language dc:language

eng

Identifier

AAI9503126
(UMI)AAI9503126

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/22184