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Protein Quality Control at the Inner Nuclear Membrane – The Asi complex in Saccharomyces cerevisiae
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Per Ljungdahl)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The nuclear envelope is a barrier comprised of outer and inner membranes that separate the cytoplasm from the nucleoplasm. The outer (ONM) and inner (INM) membranes have different physical characteristics and protein compositions. In contrast to the extensive data available on the protein quality control processes operating in the cytoplasm, endoplasmic reticulum and the nucleoplasm, the mechanisms controlling protein turnover at the INM are poorly documented. The work presented in this thesis focuses on Asi1, Asi2 and Asi3, three bona-fide integral INM proteins of the yeast Saccharomyces cerevisiae. By contrast to mammalian cells, yeast progress through the cell cycle with a closed mitosis, that is cells divide in the absence of the cyclical fragmentation/reassembly of the nuclear membrane. Consequently, examining the processes affecting the stability of the Asi proteins in yeast may provide useful paradigms for understanding the turnover of INM components in non-dividing, terminally differentiated and post-mitotic cells of metazoan origin.The results have contributed to the elucidation of the biological function of Asi1 and Asi3, which are homologous proteins with C-terminal RING domains. Asi1 and Asi3 function together as a dimeric E3 ubiquitin ligase complex that operates with ubiquitin conjugating enzymes Ubc6 and Ubc7. The Asi1/3 complex ubiquitylates transcription factors Stp1 and Stp2 when they gain inappropriate access to the nucleus in the absence of SPS-sensor activation. Intriguingly, the Asi1/3 complex also mediates the turnover of multiple membrane proteins that primarily localize to other cell membranes. This latter finding indicates that the barrier function of nuclear pore complexes is not as tight as previously thought. Consistently, asi1 null mutations are synthetic lethal when introduced into hrd1Δ ire2Δ cells with compromised ER-associated degradation (ERAD) and unfolded protein response (UPR) pathways. Together the results define Asi1/3 as components of a novel quality control pathway operating in association with the INM that acts to safeguard the identity and maintain the function of the nuclear compartment. Asi1 and Asi2 exhibit rapid turnover and their turnover is ubiquitin-dependent, exhibiting a clear requirement for Ubc7. The ubiquitylated forms of Asi1 and Asi2 are degraded by nuclear-localized proteasomes; the ubiquitylated forms exhibit enhanced stability in sts1-2 mutants. Asi1 turnover requires Cue1, the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs unimpeded in cells lacking a functional Asi1/3 complex and in cells lacking Doa10, an E3 ligase complex also known to function at the INM. Consequently, Asi1 is subject to a quality control pathway associated with INM but that is distinct from the Asi1/3 and Doa10 INM- associated degradative (INMAD) pathways. This thesis documents work that clearly demonstrates that the INM is a highly dynamic structure that possesses multiple and active quality control pathways.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2016. , 65 p.
Keyword [en]
nuclear envelope, nuclear quality control (NQC), ubiquitin, proteasome, inner nuclear membrane-associated degradation (INMAD)
National Category
Biological Sciences
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-127067ISBN: 978-91-7649-359-5 (print)OAI: oai:DiVA.org:su-127067DiVA: diva2:906004
Public defence
2016-04-19, De Geer hall, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2016-03-23 Created: 2016-02-23 Last updated: 2017-02-24Bibliographically approved
List of papers
1. Protein quality control at the inner nuclear membrane
Open this publication in new window or tab >>Protein quality control at the inner nuclear membrane
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2014 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 516, no 7531, 410-+ p.Article in journal (Refereed) Published
Abstract [en]

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression(1). The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asicomplex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer(5), we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-111393 (URN)10.1038/nature14096 (DOI)000346484800050 ()
Note

AuthorCount:16;

Available from: 2015-01-05 Created: 2015-01-02 Last updated: 2017-12-05Bibliographically approved
2. A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation
Open this publication in new window or tab >>A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation
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2014 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 16, 3603-3613 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
ERAD, Nuclear membrane, Nuclear proteasome, Protein degradation, Ubiquitylation
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-107807 (URN)10.1242/jcs.153163 (DOI)000341180000018 ()
Note

AuthorCount:5;

Available from: 2014-10-05 Created: 2014-09-29 Last updated: 2017-12-05Bibliographically approved
3. Cdc48 and Ubx1/Shp1 are components of a novel inner nuclear membrane associated degradative pathway that governs the stability of Asi1
Open this publication in new window or tab >>Cdc48 and Ubx1/Shp1 are components of a novel inner nuclear membrane associated degradative pathway that governs the stability of Asi1
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-127086 (URN)
Available from: 2016-02-24 Created: 2016-02-24 Last updated: 2016-03-10Bibliographically approved

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