Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways.
Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b(5), a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.
Borgese
N
N
Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology Center and Department of Medical Pharmacology, University of Milan, Milan, Italy. Nica@csfic.mi.cnr.it
Gazzoni
I
I
Barberi
M
M
Colombo
S
S
Pedrazzini
E
E
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011233:Precipitin Tests; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D009930:Organic Chemicals; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C107472:red dye CMXRos; D015786:Cytochromes b5; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase