Analysis of nuclear pore protein p62 glycosylation

Lubas, William A.; Smith, M.G.H.; Starr, Christopher M.; Hanover, John A.

doi:10.1021/bi00005a025

Cited by 66 publications

(49 citation statements)

References 33 publications

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“…Although the algorithms used predict that mOGT is mainly oriented toward the intermembrane space, further in vitro studies are required to confirm this localization. These findings are intriguing in light of early reports from our laboratory (Lubas et al, 1995;Lubas et al, 1997;Starr and Hanover, 1990) and the Hart laboratory (Haltiwanger et al, 1992) suggesting that both soluble and membrane-bound OGT activities were detected. This mitochondrially sequestered form of OGT (mOGT) is likely to represent the membrane-bound form of OGT reported earlier.…”

Section: Association Of Mogt With the Mitochondrial Membranementioning

confidence: 59%

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Love

Kochan

Cathey

et al. 2003

Journal of Cell Science

180

154

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O-linked GlcNAc transferase (OGT) mediates a novel glycan-dependent signaling pathway, but the intracellular targeting of OGT is poorly understood. We examined the localization of OGT by immunofluorescence microscopy, subcellular fractionation and immunoblotting using highly specific affinity-purified antisera. In addition to the expected nuclear localization,we found that OGT was highly concentrated in mitochondria. Since the mitochondrial OGT (103 kDa) was smaller than OGT found in other compartments(116 kDa) we reasoned that it was one of two predicted splice variants of OGT. The N-termini of these isoforms are unique; the shorter form contains a potential mitochondrial targeting sequence. We found that when epitope-tagged,the shorter form (mOGT; 103 kDa) concentrated in HeLa cell mitochondria,whereas the longer form (ncOGT; 116 kDa) localized to the nucleus and cytoplasm. The N-terminus of mOGT was essential for proper targeting. Although mOGT appears to be an active transferase, O-linked GlcNAc-modified substrates do not accumulate in mitochondria. Using immunoelectron microscopy and mitochondrial fractionation, we found that mOGT was tightly associated with the mitochondrial inner membrane. The differential localization of mitochondrial and nucleocytoplasmic isoforms of OGT suggests that they perform unique intracellular functions.

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Section: Association Of Mogt With the Mitochondrial Membranementioning

confidence: 59%

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Love

Kochan

Cathey

et al. 2003

Journal of Cell Science

180

154

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“…Especially noteworthy is that each Nup62 molecule, between the fingertip and N-terminal FG repeats, displays a heavily glycosylated region, each of which contains 10 single GlcNAc residues, linked to a Ser (Thr) residue (17,18). The physiological significance of what we termed the "glyco-belt" region ( Fig.…”

Section: Discussionmentioning

confidence: 98%

Ring cycle for dilating and constricting the nuclear pore

Solmaz

Blobel

Melčák

2013

Proc. Natl. Acad. Sci. U.S.A.

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We recently showed that the three "channel" nucleoporins, Nup54, Nup58, and Nup62, interact with each other through only four distinct sites and established the crystal structures of the two resulting "interactomes," Nup54•Nup58 and Nup54•Nup62. We also reported instability of the Nup54•Nup58 interactome and previously determined the atomic structure of the relevant Nup58 segment by itself, demonstrating that it forms a twofold symmetric tetramer. Here, we report the crystal structure of the relevant free Nup54 segment and show that it forms a tetrameric, helical bundle that is structurally "conditioned" for instability by a central patch of polar hydrogen-bonded residues. Integrating these data with our previously reported results, we propose a "ring cycle" for dilating and constricting the nuclear pore. In essence, three homooligomeric rings, one consisting of eight modules of Nup58 tetramers, and two, each consisting of eight modules of Nup54 tetramers, are stacked in midplane and characterize a constricted pore of 10-to 20-nm diameter. In going to the dilated state, segments of one Nup58 and two Nup54 tetrameric modules reassort into a dodecameric module, eight of which form a single, heterooligomeric midplane ring, which is flexible in a diameter range of 40-50 nm. The ring cycle would be regulated by phenylalanineglycine regions ("FG repeats") of channel nups. Akin to ligandgated channels, the dilated state of the midplane ring may be stabilized by binding of [cargo•transport-factor] complexes to FG repeats, thereby linking the ratio of constricted to dilated nuclear pores to cellular transport need.ligand gating | X-ray crystallography | nucleo-cytoplasmic transport I n evolution from prokaryotes to eukaryotes, the myriad of reactions yielding cotranscriptional and posttranscriptional assembly of ribonucleoproteins was largely confined to the nuclear compartment. This required the concomitant evolution of transport conduits of a sufficiently large diameter to allow passage of assembled ribonucleoproteins to the cytoplasm. Among these, newly assembled ribosomal subunits presented a special challenge, as they are relatively rigid bodies; the eukaryotic large ribosomal subunit, for example, has a diameter of about 30 nm. Hence, a correspondingly large nuclear pore had to evolve to accommodate the passage of ribosomal subunits, but also to protect against concomitant bidirectional leakage of the myriad of proteins, which are generally much smaller than ribonucleoproteins, to help maintain distinct nucleoplasmic and cytoplasmic proteomes. One way for meeting the challenge to transport diverse substrates of a large size range would be to endow the nuclear pore with the ability to have its central transport channel undergo dilation and constriction. However, as has been argued elsewhere (1), diameter changes in the 30-nm range might buckle the membrane, if channel proteins were directly embedded into the membrane's lipid bilayer. It could be conjectured that evolution of the nuclear pore into the massive protein...

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“…Then, we purified the labeled azidoglycoproteins via sequential anti-FLAG and immobilized metal affinity chromatographic steps and identified the captured proteins by two-dimensional liquid chromatography and tandem mass spectrometry. Importantly, numerous reported O-GlcNAcylated proteins were specifically enriched in the Ac 4 GalNAz-treated cells and absent from samples from vehicle-treated cells, including several components of the nuclear pore complex (known to be heavily glycosylated) (27,28), a Sec24 family member (29), glyceraldehyde-3-phosphate dehydrogenase (30), host cell factor C1 (31, 32), Elf-1 (33), and OGT itself (34,35) (Figs. S5B and S6).…”

Section: A Pilot Proteomics Experiments Demonstrates the Utility Of Acmentioning

confidence: 99%

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

Boyce

Carrico

Ganguli

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

299

325

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Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

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Analysis of nuclear pore protein p62 glycosylation

Cited by 66 publications

References 33 publications

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

Ring cycle for dilating and constricting the nuclear pore

Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway

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