The N terminus of microsomal delta 9 stearoyl-CoA desaturase contains the sequence determinant for its rapid degradation.
Stearoyl-CoA desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a model for selective degradation of short-lived proteins of the endoplasmic reticulum (ER). The mechanism whereby specific ER proteins are targeted for degradation in the midst of stable proteins coexisting in the same membrane is unknown. To investigate the intracellular fate of SCD and to identify the determinants involved in the rapid turnover of SCD, we created chimeras of SCD tagged at the C terminus with the green fluorescent protein (GFP). The fusion proteins were expressed in Chinese hamster ovary cells and exhibited an ER localization. Unlike native GFP, the SCD-GFP construct was unstable and had a half life of a few hours. Truncated fusion proteins consisting of residues 27-358 and 45-358 of SCD linked to the N terminus of GFP were stable. To investigate the general applicability of the N terminus of SCD in the destabilization of proteins, we fused residues 1-33 of SCD to the N terminus of GFP. The resulting chimera was extremely short lived. To investigate the effect of membrane sidedness on the fusion protein degradation, we attached a lumenal targeting signal to the N terminus of SCD 1-33-GFP. The construct was localized to the lumen of ER and was metabolically stable, indicating that SCD degradation signal functions on the cytosolic rather than the lumenal side of the ER. These results demonstrate that the N-terminal segment of some 30 residues of SCD constitutes a motif responsible for the rapid degradation of SCD.
Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06030-3305, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D006868:Hydrolysis; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013230:Stearoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D013230:Stearoyl-CoA Desaturase