An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

Abstract

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 × 10(−5) M. ATP transport into ER vesicles was specifically inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3′‐O‐(4‐benzoyl)benzoyl‐ATP (Bz2‐ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2‐ATP blocked protein translocation in vitro measured co‐ as well as post‐translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS‐treated membranes. These results demonstrate that translocation requires a DIDS and Bz2‐ATP‐sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.

O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

“…Wild-type alpha-factor precursor is N-glycosylated at three sites in its proregion (Kurjan and Herskowitz, 1982). Conversion of the asparagine residues in the N-glycosyl acceptor sites to glutamine residues results in a protein that is recognized in the ER as misfolded and subsequently transported to the cytosol for disposal by the proteasomes (Mayinger and Meyer, 1993;McCracken and Brodsky, 1996). Most serine and threonine residues in alpha-factor precursor that could serve as Omannosyl acceptors are clustered around the N-glycosylation sites (Kurjan and Herskowitz, 1982).…”

“…We therefore asked whether misfolded proteins in the ER acquire a covalent modification that serves as an export signal during prolonged residence in the ER lumen. As substrate proteins we used [ 35 S]methionine-labeled, in vitro translated wildtype alpha-factor precursor (pp␣f) or a mutant counterpart in which the three N-glycosylation acceptor sites in the proregion had been destroyed by site-directed mutagenesis (p⌬gp␣f) (Mayinger and Meyer, 1993;McCracken and Brodsky, 1996). In the presence of ATP and an ATP-regenerating system, both proteins can be translocated into yeast microsomes posttranslationally, resulting in signal cleaved mutant alpha-factor precursor (⌬gp␣f) and signal-cleaved, triply Nglycosylated wild-type alpha-factor precursor (3gp␣f), respectively.…”

“…As a substrate we used a mutant form of the yeast pheromone precursor prepro alpha-factor, which had its N-glycosylation acceptor sites removed by site-directed mutagenesis (Mayinger and Meyer, 1993). The N-glycosylation site mutant alpha-factor precursor protein (⌬gp␣f) is subject to ERassociated degradation in vivo and in a cell-free assay based on yeast microsomes and cytosol (McCracken and Brodsky, 1996).…”

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

“…We asked whether ATP was required in the ER lumen. We performed peptide export experiments in the presence of the anion transport inhibitor DIDS, a stilbene derivative that inhibits anion transporters and interferes with transport of ATP into the ER lumen (30). As illustrated in Fig.…”

Reconstitution, Identification, and Purification of the Rat Liver Golgi Membrane GDP-fucose Transporter

“…The reaction mixture was then applied to a 3-ml Dowex 1 ϫ 2-100 column (Sigma) as described previously (19). Fractions of 0.35 ml were collected, and the radioactivity was determined by liquid scintillation spectrometry.…”