Endoplasmic reticulum retention determinants in the transmembrane and linker domains of cytochrome P450 2C1.
The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.
Szczesna-Skorupa
E
E
Departments of Molecular and Integrative Physiology and Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Kemper
B
B
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D021382:Protein Sorting Signals; D003577:Cytochrome P-450 Enzyme System; C096409:CYP2C1 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2