Alg13p, the Catalytic Subunit of the Endoplasmic Reticulum UDP-GlcNAc Glycosyltransferase, Is a Target for Proteasomal Degradation

Averbeck, Nicole; Gao, Xiao‐Dong; Nishimura, Shin‐Ichiro; Dean, Neta

doi:10.1091/mbc.e07-10-1077

Cited by 19 publications

(17 citation statements)

References 39 publications

(51 reference statements)

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“…ALG14 is highly conserved among species and contains a large C‐terminal cytosolic tail which binds to ALG13 and an N terminus that resides within the ER lumen . The protein is necessary for the ER localization and proper function of ALG13 as cytosolic ALG13 is very short‐lived and rapidly undergoes proteasomal degradation …”

Section: Discussioncontrasting

confidence: 97%

Genomic screening in rare disorders: New mutations and phenotypes, highlighting ALG14 as a novel cause of severe intellectual disability

et al. 2018

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We have investigated 20 consanguineous families with multiple children affected by rare disorders. Detailed clinical examinations, exome sequencing of affected as well as unaffected family members and further validation of likely pathogenic variants were performed. In 16/20 families, we identified pathogenic variants in autosomal recessive disease genes (ALMS1, PIGT, FLVCR2, TFG, CYP7B1, ALG14, EXOSC3, MEGF10, ASAH1, WDR62, ASPM, PNPO, ERCC5, KIAA1109, RIPK4, MAN1B1). A number of these genes have only rarely been reported previously and our findings thus confirm them as disease genes, further delineate the associated phenotypes and expand the mutation spectrum with reports of novel variants. We highlight the findings in two affected siblings with splice altering variants in ALG14 and propose a new clinical entity, which includes severe intellectual disability, epilepsy, behavioral problems and mild dysmorphic features, caused by biallelic variants in ALG14.

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Section: Discussioncontrasting

confidence: 97%

Genomic screening in rare disorders: New mutations and phenotypes, highlighting ALG14 as a novel cause of severe intellectual disability

et al. 2018

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“…Protein concentrations of lysates were determined by the Bradford method. Samples (20 g) were fractionated by SDS-PAGE (12%) and immunoblotted, as described previously (2). yEmRFP protein levels were quantitated by immunoblotting with anti-FLAG antibodies (Sigma) diluted 1: 2,000 to detect FLAG-tagged yEmRFP expressed at a single copy or multiple copies (Fig.…”

Section: Methodsmentioning

confidence: 99%

Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

Keppler-Ross¹,

Douglas

Konopka

et al. 2010

Eukaryot Cell

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The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.Candida albicans is an opportunistic fungus that normally resides in the human gut (26) and can cause mucosal infections. When host immune defenses are compromised or when anatomical breaches permit extreme fungal burdens, systemic and often lethal fungal colonization throughout the body can occur. In hospital-acquired bloodstream infections, the rate of mortality, hospital cost, and length of stay associated with disseminated candidiasis now outrank those associated with bacterial infections (37, 43). The most effective host barrier that limits Candida infections is microbial destruction by phagocytic cells of the innate immune system. In a healthy host, phagocytes-macrophages, neutrophils, and dendritic cells-recognize, ingest, and destroy the invading yeast by phagocytosis.The first step of a fungal infection requires the recognition of yeast by phagocytes. Despite the medical importance of this reaction, it remains poorly understood. As the interface between the yeast and its host, the fungal cell wall is crucial for recognition. The wall is a complex structure consisting of an elastic network of polysaccharides (glucans and chitin) that surrounds the plasma membrane and that in most yeast and fungi contains many diffe...

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“…Formation of Alg13/14 complex has also been suggested to be a target for regulation of N-linked glycosylation. Unassembled excess cytosolic Alg13p inhibits N-linked glycosylation, and is prevented to accumulate intracellularly by proteasomal degradation (10), providing further evidence for the importance of Alg13/14 complex formation.…”

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confidence: 87%

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

Gao

Moriyama

Miura

et al. 2008

Journal of Biological Chemistry

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The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal ␣-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal ␣-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal ␣-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal ␤-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.

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Alg13p, the Catalytic Subunit of the Endoplasmic Reticulum UDP-GlcNAc Glycosyltransferase, Is a Target for Proteasomal Degradation

Cited by 19 publications

References 39 publications

Genomic screening in rare disorders: New mutations and phenotypes, highlighting ALG14 as a novel cause of severe intellectual disability

Genomic screening in rare disorders: New mutations and phenotypes, highlighting ALG14 as a novel cause of severe intellectual disability

Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

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