The N-terminus of retinol dehydrogenase type 1 signals cytosolic orientation in the microsomal membrane.
We determined the orientation of the SDR (short-chain dehydrogenase/reductase) rat RoDH1 (retinol dehydrogenase type 1) in the endoplasmic reticulum to provide insight into its function in retinol metabolism, and to resolve whether retinoid-metabolizing SDRs differ from several other SDRs by requiring a C-terminal segment for the membrane orientation. In contrast to several soluble SDRs, the membrane-associated RoDH1 has hydrophobic extensions N- and C-terminal to the SDR core. Confocal microscopy and/or proteinase K protection assays of RoDH1, RoDH1 mutants, and RoDH1-green fluorescent protein fusion proteins showed that the N-terminal segment anchors RoDH1 to the endoplasmic reticulum membrane facing the cytosol. The C-terminal hydrophobic segment increases the relative proportion of RoDH1 associated with the endoplasmic reticulum, but has no affect on orientation. Deletion of either or both extensions causes nearly total loss of enzyme activity, possibly through altering the nature of RoDH1 association with membranes, or destabilizing the enzyme, but does not alter the expression of RoDH1 or convert it into a soluble protein. The latter suggests that the SDR core of RoDH1 has marked external hydrophobicity that causes nonspecific membrane association.
Wang
J
J
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 97420-3104, USA.
Bongianni
J K
JK
Napoli
J L
JL
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000072467:Cytochrome P450 Family 2; D003600:Cytosol / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D014801:Vitamin A / Q000737:chemistry
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014801:Vitamin A; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2