Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins

Fujimuro, Masahiro; Sawada, Hitoshi; Yokosawa, Hideyoshi

doi:10.1016/0014-5793(94)00647-4

Cited by 237 publications

(222 citation statements)

References 43 publications

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“…The FK1 and FK2 antibodies specifically recognize ubiquitin-conjugated proteins, but not unconjugated ubiquitin (FK1 labels poly-ubiquitylated conjugates, FK2 labels mono-and poly-ubiquitylated proteins). 55,56 As shown in Figures 1b' and c', the immunoreactivity of FK1 and FK2 antibodies increases in prosβ2 mutant cells. We also find increased FK1 and FK2 labeling in Mov34 mutant cells (Supplementary Figure S1).…”

Section: Resultsmentioning

confidence: 77%

The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

et al. 2016

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A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired.

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Section: Resultsmentioning

confidence: 77%

The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

et al. 2016

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“…It is involved in ERAD, wherein proteins are extracted from the ER, ubiquitinated, and then degraded by proteasomes (reviewed in Tsai et al, 2002). To analyze the disposition of ubiquitinated proteins in cells expressing p97(E578Q), we used an antibody that recognizes ubiquitin conjugates (Fujimuro et al, 1994; Figure 7A). This showed that expressing p97(E578Q) caused a significant build-up of ubiquitinated proteins on intracellular membranes.…”

Section: P97(e578q) Promotes Accumulation Of Ubiquitin Conjugates On mentioning

confidence: 99%

Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

Dalal

Rosser

Cyr

et al. 2004

MBoC

166

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NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATPhydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion. INTRODUCTIONMembrane fusion is an essential step in all forms of vesicle trafficking and organelle assembly. Fusion is driven by a series of regulated protein-protein interactions. Many participating proteins have been identified, and their specific roles are gradually coming to light (reviewed in Jahn et al., 2003). Among these proteins are a number of ATPases.N-ethyl maleimide sensitive factor (NSF) was one of the first proteins specifically linked to membrane fusion (Wilson et al., 1989). It belongs to a family of chaperone-like ATPases known as AAA (ATPases associated with a variety of cellular activities) proteins (Neuwald et al., 1999). NSF together with ␣-SNAP (soluble NSF attachment protein) dissociates the SNARE (SNAP receptor) complexes that promote association and fusion of cellular membranes. More recently, NSF has also been implicated in other cellular processes on the basis of its ability to bind the AMPA receptor GluR2, ␤-arrestin 1, and GATE-16 (reviewed in Whiteheart et al., 2001). However, its role in SNARE disassembly and membrane fusion remains its best understood function.Not all ATP-requiring steps that lead to membrane fusion can be attributed to NSF (Goda and Pfeffer, 1991;Latterich and Schekman, 1994;Rodriguez et al., 1994;Wilson, 1995).Other ATPases must therefore be involved. One AAA protein thought to be an alternate to NSF is known as p97 (also referred to as valosin-containing protein, VCP). p97 is an abundant and highly conserved protein (Peters et al., 1990) whose cellular function has been the subject of much debate. A break in the mystery of p97's function came when it turn...

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“…For this experiment, we used the FK2 antibody, which specifically detects ubiquitin moieties on cargo proteins (Fujimuro et al, 1994). When wild-type AMSH was expressed, only a marginal amount of ubiquitinated cargo was observed (Fig.…”

Section: Amsh Associates With the Escrt-iii Protein Chmp3mentioning

confidence: 99%

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

Kyuuma

Kikuchi

Kojima

et al. 2006

Cell Struct. Funct.

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and 6 These two authors contributed equally to this work.ABSTRACT. The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.

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Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins

Cited by 237 publications

References 43 publications

The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

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