Abstract
The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.
Highlights
In parallel, protein folding/assembly in the endoplasmic reticulum (ER) relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER
We first asked whether the hydrophobic segment at the N terminus of Mnl1p functions as a cleavable signal sequence or a transmembrane segment to anchor the protein to the ER membrane
In the L614A mutant, which could partially form the C1–C3 disulfide bond, the C1–C3 disulfide bond was more susceptible to reduction by DTT treatment than in wild type Mnl1p-FLAG (Fig. 5C, lane 28, ϪME). Some of those mutants exhibited high molecular weight bands (Fig. 5C, lanes 25, 30, and 31, ϪME), they were not detected with anti-protein-disulfide isomerase (PDI) antibodies, suggesting that they are aggregated forms of the mutant Mnl1p. These results collectively suggest that the conserved amino acid residues in the C-terminal domain including Asp607, Leu614, Glu627, and Trp636 are critical for stable interactions with PDI, which is essential for correct disulfide-bond formation between C1 and C3 in the mannosidase homology domain (MHD) of Mnl1p, it is not clear whether these residues are directly involved in the interactions with PDI or affect folding of the C-terminal domain
Summary
Protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. The 250 –300-kDa bands were detected by antiPDI antibodies under nonreducing conditions (Fig. 2E, lanes 6 and 8), suggesting that the 250 –300-kDa bands correspond to complexes formed by intermolecular disulfide bond(s) between Mnl1p and PDI.
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