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., Villa, A., Longhi, R., Bulbarelli, A., & Borgese, N. (2000). Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum. THE JOURNAL OF CELL BIOLOGY, 148(5), 899-914 [10.1083/jcb.148.5.899].

Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum

Villa, A;Bulbarelli, A
Membro del Collaboration Group
;
2000

Abstract

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.
Articolo in rivista - Articolo scientifico
Scientifica
Glycosylation; Protein Structure, Tertiary; Cell Line; Dogs; Cell Compartmentation; Endoplasmic Reticulum; Golgi Apparatus; Cricetinae; Galactose; Precipitin Tests; Protein Processing, Post-Translational; Animals; Rabbits; Blotting, Western; Binding Sites; Intracellular Membranes; Acetylgalactosamine; Enzyme Inhibitors; Protein Synthesis Inhibitors; Mannosidases; Molecular Sequence Data; Cytochromes b5; Amino Acid Sequence; Protein Binding
English
Pedrazzini, E., Villa, A., Longhi, R., Bulbarelli, A., & Borgese, N. (2000). Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum. THE JOURNAL OF CELL BIOLOGY, 148(5), 899-914 [10.1083/jcb.148.5.899].
Pedrazzini, E; Villa, A; Longhi, R; Bulbarelli, A; Borgese, N
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/19406
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