Integration of Cytochrome b5 into Endoplasmic Reticulum Membrane: Participation of Carboxy-Terminal Portion of the Transmembrane Domain

Abstract

Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…These and other results confirm the importance of the conserved residues in the -R/H-x-Y/F-motif and that this sequence is a basis for predicting the localization of Cb5 proteins to the ER in plant cells. Overall, the role of the tail sequences in ER Cb5s is likely related to increasing ER targeting efficiency or, as reported recently for the rat ER Cb5 isoform (Tanaka et al, 2003), facilitating membrane integration and assembly. It is also conceivable that the more complex targeting signals (i.e., distinct targeting elements in both the TMD and tail) of plant Cb5 proteins, as described above, have evolved to allow for better discrimination between ER, mitochondrial, and, unique to plant cells, plastid targeting pathways and that these signals provide higher fidelity/specificity with the receptor machinery of the various organelles that can acquire TA membrane proteins.…”

Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal

“…It is unknown whether it represents P450 synthesized on free polysomes, waiting [like cytochrome b 5 (b 5 ) and b 5 - reductase (Borgese and Meldolesi, 1980; Okada et al, 1982; Tanaka et al, 2003)] to be incorporated posttranslationally into the ER-membranes. Alternatively, in extrahepatic tissues, cytosolic CYP1A1 may have some unknown function or monitored in transit on its way to another organelle and/or disposal.…”