Dimeric organization of the yeast oligosaccharyl transferase complex

Chavan, Manasi; Chen, Zhiqiang; Li, Guangtao; Schindelin, Hermann; Lennarz, William J.; Li, Huilin

doi:10.1073/pnas.0603262103

Cited by 37 publications

(22 citation statements)

References 55 publications

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“…This positioning of OST in the native translocon allows for concomitant scanning of a nascent polypeptide for glycosylation sites, while it is conducted into the ER lumen, thus providing a basic understanding of how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. From the good agreement of the approximate molecular weight of LD (170-200 kDa) with the lumenal mass of OST, as well as the absence of multiply-occurring structural features in LD, we furthermore conclude that OST is present in only one copy in the native translocon, in contrast to previous hypotheses of a dimeric OST organization 11 .…”

Section: Resultscontrasting

confidence: 52%

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

et al. 2014

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In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the proteinconducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

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Section: Resultscontrasting

confidence: 52%

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

et al. 2014

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“…gp78 has a multifunctional cytosolic tail that conveys E3 activity, binds polyubiquitin, and recruits Ubc7 and p97/VCP (17,18,35). Recent evidence indicates that p97/ VCP recruited by gp78 is associated with peptide:N-glycanase (39,42). We have now demonstrated that gp78 has a p97/VIM responsible for recruiting p97/VCP to the ER.…”

Section: Discussionmentioning

confidence: 70%

The Role of a Novel p97/Valosin-containing Protein-interacting Motif of gp78 in Endoplasmic Reticulum-associated Degradation

Ballar

Shen

Yang

et al. 2006

Journal of Biological Chemistry

118

155

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Improperly folded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation, a process that dislocates misfolded proteins from the ER membrane into the cytosol, where they undergo proteasomal degradation. Dislocation requires a subclass of ubiquitin ligases that includes gp78 in addition to the AAA ATPase p97/VCP and its cofactor, the Ufd1-Npl4 dimer. We have previously reported that gp78 interacts directly with p97/VCP. Here, we identify a novel p97/ VCP-interacting motif (VIM) within gp78 that mediates this interaction. We demonstrate that the VIM of gp78 recruits p97/ VCP to the ER, but has no effect on Ufd1 localization. We also show that gp78 VIM interacts with the ND1 domain of p97/VCP that was shown previously to be the binding site for Ufd1. To evaluate the role of Ufd1 in gp78-p97/VCP-mediated degradation of CD3␦, a known substrate of gp78, RNA interference was used to silence the expression of Ufd1 and p97/VCP. Inhibition of p97/VCP, but not Ufd1, stabilized CD3␦ in cells that overexpress gp78. However, both p97/VCP and Ufd1 appear to be required for CD3␦ degradation in cells expressing physiological levels of gp78. These results raise the possibility that Ufd1 and gp78 may bind p97/VCP in a mutually exclusive manner and suggest that gp78 might act in a Ufd1-independent degradation pathway for misfolded ER proteins, which operates in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.

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“…The C terminus of DC2 is weakly homologous, sharing less than 10 and 18% homology with the yeast subunits, Ost3p and Ost6p, respectively. It is believed that Ost3/6p are present in two subcomplexes that associate either with Sbh1p or Sbh2p, respectively, forming two structurally different translocons (43,44). Recently, Ost3/6p have been shown to contain thioredoxin-like folds, which would enable the oxidation folding of glycoproteins (45).…”

Section: Discussionmentioning

confidence: 99%

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

Wilson

Magnaudeix

Yardin

et al. 2011

Journal of Biological Chemistry

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The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPP␣ secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the ␥-secretase complex, resulting in a reduction of the PS1 active fragment blocking A␤ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active ␥-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of A␤ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the ␥-secretase and affect the APP processing pathway. Alzheimer disease (AD)2 is the primary cause of adult onset dementia, with a dramatic increase in the incidence of AD apparent in our aging population. AD is pathologically characterized by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-␤ (A␤) peptides, A␤40 and A␤42 (1). The early onset familial form of AD is linked to three genes, amyloid precursor protein (APP) and presenilin (PS1 and PS2) (2, 3), strongly suggesting that the production of A␤ is a key factor in the pathogenesis of AD. A␤ is generated by proteolysis of APP, driven by the secretases found in the cell. Prior to proteolysis, APP undergoes a number of post-translational modifications, including N-glycosylation in the endoplasmic reticulum (ER) and O-glycosylation in the Golgi apparatus. In order to generate A␤40 and A␤42, APP is first cleaved by ␤-secretase and then by ␥-secretase. For the cleavage of APP, ␤-secretase competes with ␣-secretase, which produces non-amyloidogenic peptides (4). ␥-Secretase is an aspartyl protease complex composed of four core components, including presenilins (PS1/PS2), presenilin enhancer 2 (PEN2), nicastrin, and anterior pharynx-defective 1 (APH1) (2). Presenilin is the catalytic core of the ␥-secretase complex consisting of nine transmembrane domains (5) and is cleaved by an unknown protease called "presenilinase" or self-cleavage stimulated by PEN2 binding of the cytosolic loop between transmembrane domains 6 and 7, releasing N-and C-terminal PS1 fragments, th...

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Dimeric organization of the yeast oligosaccharyl transferase complex

Cited by 37 publications

References 55 publications

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

The Role of a Novel p97/Valosin-containing Protein-interacting Motif of gp78 in Endoplasmic Reticulum-associated Degradation

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex, Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

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