Two distinctly localized p-type ATPases collaborate to maintain organelle homeostasis required for glycoprotein processing and quality control.

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.

Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism.

BACKGROUND: Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene. RESULTS: After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase. CONCLUSION: Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity.

Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD.

The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.

Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol.

N-Glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Here we identify and characterize two essential yeast proteins having homology to bacterial glycosyltransferases, designated Alg13p and Alg14p, as being required for the formation of GlcNAc(2)-PP-dolichol (Dol), the second step in the biosynthesis of the unique lipid-linked core oligosaccharide. Down-regulation of each gene led to a defect in protein N-glycosylation and an accumulation of GlcNAc(1)-PP-Dol in vivo as revealed by metabolic labeling with [(3)H]glucosamine. Microsomal membranes from cells repressed for ALG13 or ALG14, as well as detergent-solubilized extracts thereof, were unable to catalyze the transfer of N-acetylglucosamine from UDP-GlcNAc to [(14)C]GlcNAc(1)-PP-Dol, but did not impair the formation of GlcNAc(1)-PP-Dol or GlcNAc-GPI. Immunoprecipitating Alg13p from solubilized extracts resulted in the formation of GlcNAc(2)-PP-Dol but required Alg14p for activity, because an Alg13p immunoprecipitate obtained from cells in which ALG14 was down-regulated lacked this activity. In Western blot analysis it was demonstrated that Alg13p, for which no well defined transmembrane segment has been predicted, localizes both to the membrane and cytosol; the latter form, however, is enzymatically inactive. In contrast, Alg14p is exclusively membrane-bound. Repression of the ALG14 gene causes a depletion of Alg13p from the membrane. By affinity chromatography on IgG-Sepharose using Alg14-ZZ as bait, we demonstrate that Alg13-myc co-fractionates with Alg14-ZZ. The data suggest that Alg13p associates with Alg14p to a complex forming the active transferase catalyzing the biosynthesis of GlcNAc(2)-PP-Dol.

Major histocompatibility complex class I molecules expressed with monoglucosylated N-linked glycans bind calreticulin independently of their assembly status.

The assembly of major histocompatibility complex (MHC) class I molecules with peptides in the endoplasmic reticulum (ER) is a critical step in the presentation of viral antigens to CD8+ T cells. This process is subject to quality control restrictions that prevent free class I heavy chains (HCs) and peptide-free HC-beta(2)-microglobulin (beta(2)m) dimers from exiting the ER. The lectin-like chaperone calreticulin associates with HC-beta(2)m heterodimers prior to peptide binding, but its precise role in regulating the subsequent events of peptide association and ER to Golgi transport remains undefined. In vitro analysis of the assembly process has been limited by the specificity of calreticulin for monoglucosylated N-linked glycans, which are transient biosynthetic intermediates. To address this problem, we developed a novel expression system using Saccharomyces cerevisiae glycosylation mutants to produce class I HC bearing N-linked oligosaccharides with the specific structure Glc(1)Man(9)GlcNAc(2). The monoglucosylated glycan proved to be both necessary and sufficient for in vitro binding of calreticulin to MHC class I molecules. Calreticulin bound as efficiently to peptide-loaded MHC class I complexes as it did to folding intermediates created in vitro, namely free class I HC and empty HC-beta(2)m heterodimers. Thus, calreticulin is unable to discriminate between native and non-native MHC class I conformations and therefore unlikely to play a role in the recognition and release of peptide-loaded complexes from the ER. Furthermore, the recombinant expression system developed in this study can be used to produce a broad range of calreticulin substrates to elucidate its general mechanism of activity in vitro.