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© The Rockefeller University Press, 0021-9525/2000//899 $5.00
The Journal of Cell Biology, Volume 148, Number 5, , 2000 899-914

Original Article

Mechanism of Residence of Cytochrome B(5), a Tail-Anchored Protein, in the Endoplasmic Reticulum



Emanuela Pedrazzinia, Antonello Villaa,b, Renato Longhic, Alessandra Bulbarellia, and Nica Borgesea,d

a Consiglio Nazionale Ricerche Cellular and Molecular Pharmacology Center, Department of Pharmacology, University of Milan, Italy 20129
b Biological and Technological Research Department, Scientific Institute San Raffaele, Milan, Italy 20132
c Consiglio Nazionale Ricerche Institute of Biocatalysis and Molecular Recognition, Milan, Italy 20133
d Faculty of Pharmacy, University of Catanzaro "Magna Graecia", Roccelletta di Borgia (Catanzaro), Italy 88021
Consiglio Nazionale Ricerche Cellular and Molecular Pharmacology Center, via Vanvitelli 32, 20129 Milan, Italy.39-02-7490-57439-02-7014-6250

nica{at}farma.csfic.mi.cnr.it

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.

Key Words: cell compartmentation • endoplasmic reticulum • glycosylation • Golgi apparatus • membrane proteins

© 2000 The Rockefeller University Press

Abbreviations used in this paper: Ab, antibody; b(5), cytochrome b(5); BFA, brefeldin A; CHX, cycloheximide; Endo, endoglycosidase; GalNac, N-acetylgalactosamine; GlcNac, N-acetylglucosamine; GlcNH2, glucosamine; IC, intermediate compartment; OKA, okadaic acid; PDI, protein disulfide isomerase; PNS, postnuclear supernatant(s); SLO, streptolysin O; TA, tail-anchored; TMD, transmembrane domain; wt, wild-type.


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