| 1. |
- Boban, Mirta, et al.
(författare)
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A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation
- 2014
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:16, s. 3603-3613
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.
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| 2. |
- Boban, Mirta, et al.
(författare)
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Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:4, s. 2489-2495
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Tidskriftsartikel (refereegranskat)abstract
- Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.
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| 3. |
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| 4. |
- Boban, Mirta
(författare)
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Transcriptional regulation by inner nuclear membrane proteins
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All cells sense discrete environmental signals and respond by making appropriate adjustments in patterns of gene expression. To achieve this, signaling pathways translate information received at the cell surface into a transcriptional response. An essential feature of inducible signal transduction pathways is the maintenance of the off state of the gene expression when inducing signals are absent. This thesis addresses the mechanisms that ensure the repressed state of SPS-sensor regulated genes in the yeast Saccharomyces cerevisiae. Yeast cells use the plasma membrane localized SPS-sensor to sense extracellular amino acids present in their growth environment. In response to amino acids, the SPS-sensor endoproteolytically activates Stp1 and Stp2, two latent cytoplasmic transcription factors with redundant functions. SPS-sensor mediated processing removes cytoplasmic retention motifs within the N-terminal domains of these factors. The shorter activated forms efficiently target to the nucleus, bind promoters of SPS-sensor regulated genes and induce their transcription. Recessive loss-of-function mutations in ASI1, ASI2 and ASI3 (amino acid sensor independent) genes bypass the requirement of a functional SPS-sensor and constitutively induce SPS-sensor regulated gene expression. The recessive nature of the asi mutations suggested that Asi proteins act to negatively regulate SPS-sensor signaling. Interestingly, all three Asi proteins are integral membrane proteins with multiple membrane-spanning segments. The goal of this thesis has been to elucidate the function of Asi1, Asi2 and Asi3. Genetic and biochemical tests demonstrate that Asi proteins are bona fide constituents of the SPS-sensing pathway. All three Asi proteins localize to the inner nuclear membrane where they function in concert to maintain the latent properties of Stp1 and Stp2 under non-inducing conditions. The data indicate that cytoplasmic retention mechanisms, which prevent nuclear accumulation of latent forms of Stp1 and Stp2, are not completely efficient, and low levels of fulllength forms of Stp1 and Stp2 are able to enter the nucleus. In cells lacking either of the Asi proteins, the unprocessed forms of Stp1 and Stp2 that enter the nucleus are able to bind SPSsensor dependent promoters and inappropriately induce gene expression. Thus, the Asi proteins are required to ensure the repressed state of SPS-sensor signaling in the absence of inducing amino acids by restricting promoter access of full length Stp1 and Stp2. The nuclear factor Dal81 was found to augment the activation potential of Stp1 and Stp2 by enhancing their efficiency to bind SPS-sensor regulated promoters. Thus, Dal81 greatly enhances the sensitivity of the SPS sensing pathway. Strikingly, in cells lacking Dal81, the negative regulatory activity of Asi1, Asi2 and Asi3 is not required to maintain the "off state of SPS-sensor regulated gene expression. In summary, this thesis documents the discovery of a novel role of inner nuclear membrane proteins and illuminate an additional and unanticipated layer of transcriptional control in eukaryotic cells.
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| 5. |
- Pantazopoulou, Marina, et al.
(författare)
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Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation
- 2016
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 129:20, s. 3770-3780
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t(1/2)<= 30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.
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| 6. |
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