Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum.
Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two. Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretory pathway by largely unknown mechanisms. Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins. We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms. Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation. These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.
Pedrazzini
E
E
Consiglio Nazionale Ricerche Cellular and Molecular Pharmacology Center, Department of Pharmacology, University of Milan, Italy.
Villa
A
A
Longhi
R
R
Bulbarelli
A
A
Borgese
N
N
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits
D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D015786:Cytochromes b5; D008361:Mannosidases; D000116:Acetylgalactosamine; D005690:Galactose