Abstract
C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc). Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells. The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation. Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation. The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter. Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc. Similarly, only low ATP concentrations (below 1 microm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.
Highlights
C-tail-anchored proteins (TA1 proteins) constitute a class of integral membrane proteins that are held in the phospholipid bilayer by a single segment of hydrophobic amino acids close to
A point that remained controversial for many years was whether TA proteins are bona fide transmembrane proteins, with the C-terminal residues exposed at the exoplasmic side of the membrane, or rather have a hairpin configuration with the hydrophobic stretch looping back to the cytosolic face of the bilayer
In recent years it has become clear that TA proteins can translocate their C-terminal residues across the endoplasmic reticulum (ER) membrane
Summary
C-tail-anchored proteins (TA1 proteins) constitute a class of integral membrane proteins that are held in the phospholipid bilayer by a single segment of hydrophobic amino acids close to. 1 and 2 for reviews), and control of gene expression [3] Consistent with these different functions, TA proteins are found on a variety of membranes, such as those of the endoplasmic reticulum (ER) and of the Golgi complex, the plasma membrane and the mitochondrial outer membrane. A point that remained controversial for many years was whether TA proteins are bona fide transmembrane proteins, with the C-terminal residues exposed at the exoplasmic side of the membrane, or rather have a hairpin configuration with the hydrophobic stretch looping back to the cytosolic face of the bilayer. Using glycosylation as a criterion for translocation, a variety of different C-terminal sequences, deriving from cytosolic or exoplasmic portions of different proteins, have been shown to be translocated, indicating that the translocation process is not very selective with regard to the residues that are transferred to the ER lumen
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