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Membrane-bound and soluble proteins of the secretory pathway are commonly glycosylated in the endoplasmic reticulum. These adducts have many biological functions, including, notably, their contribution to the maturation of glycoproteins. N-linked glycans are of oligomeric structure, forming configurations that provide blueprints to precisely instruct the folding of protein substrates and the quality control systems that scrutinize it. O-linked mannoses are simpler in structure and were recently found to have distinct functions in protein quality control that do not require the complex structure of N-linked glycans. Together, recent studies reveal the breadth and sophistication of the roles of these glycan-directed modifications in protein biogenesis.

Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man(8)GlcNAc(2) carbohydrates bound to unfolded proteins, yielding Man(7)GlcNAc(2). This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum (ER) quality control mechanisms monitor the folding of nascent secretory and membrane polypeptides. Immature molecules are actively retained in the folding compartment whereas proteins that fail to fold are diverted to proteasome-dependent degradation pathways. We report that a key pathway of ER quality control consists of a two-lectin receptor system consisting of Yos9p and Htm1/Mnl1p that recognizes N-linked glycan signals embedded in substrates. This pathway recognizes lumenally oriented determinants of soluble and membrane proteins. Yos9p binds directly to substrates to discriminate misfolded from folded proteins. Substrates displaying cytosolic determinants can be degraded independently of this system. Our studies show that mechanistically divergent systems collaborate to guard against passage and accumulation of misfolded proteins in the secretory pathway.

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1 (control of HMG-CoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role. COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca(2+)/Mn(2+) pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside.