| dc.contributor.advisor | Stein, Alexander Dr. | |
| dc.contributor.author | Vasic, Vedran | |
| dc.date.accessioned | 2020-03-11T09:36:56Z | |
| dc.date.available | 2020-06-25T22:50:02Z | |
| dc.date.issued | 2020-03-11 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-135C-F | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7915 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Reconstitution of retrotranslocation by the Hrd1 ubiquitin ligase with purified components | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Rehling, Peter Prof. Dr. | |
| dc.date.examination | 2019-06-27 | |
| dc.description.abstracteng | In eukaryotic organisms, a large fraction of newly-synthesized proteins are destined for
the endomembrane system or for secretion. The entry gate for these proteins is the
endoplasmic reticulum (ER). Proteins are imported into the ER in an unfolded state,
where they fold and assemble into their native conformations before being exported to
the Golgi apparatus. Proteins which fail to fold or assemble correctly have a propensity
to aggregate in the ER, which is toxic for the cell. Accordingly, a quality control pathway
termed ER-associated protein degradation (ERAD) recognizes misfolded proteins
and retrotranslocates them across the ER membrane into the cytosol, where they are
ubiquitinated and degraded by the proteasome.
One of the biggest questions about ERAD is how substrates are retrotranslocated
across the ER membrane. Recent evidence implicates one of the central components
of ERAD, the Hrd1 ubiquitin ligase, in forming a retrotranslocon. Despite this, it is
still unknown if Hrd1 is sufficient for retrotranslocation of luminal ERAD substrates.
I created a novel reconstituted system to study retrotranslocation, in which luminal
substrates are encapsulated into liposomes and delivered to the luminal side of Hrd1
by SNARE-mediated fusion. The encapsulation was efficient and fusion was shown
to mix membrane proteins and luminal contents between liposomes, a technique that
has broad applications in membrane protein research. However, retrotranslocation by
Hrd1 could not be detected after fusion, which was most likely because of substrate
aggregation.
In another set of reconstitutions, Hrd1 reconstituted in nanodiscs bound misfolded
proteins on its luminal side, while Hrd1 reconstituted in liposomes bound misfolded
proteins with high affinity on its cytosolic side upon autoubiquitination. This was
dependent on autoubiquitination in the RING domain. Misfolded proteins could be
partially released from the cytoplasmic binding site by deubiquitination. Hrd1 was also
incorporated into planar lipid bilayers, and was shown to have channel activity and to
conduct ions in a voltage-dependent manner. This was dependent on autoubiquitination
in its RING domain. Substrate addition stimulated channel gating and opened
the pore to diameters sufficient to fit multiple alpha helices. Remarkably, deubiquitination
of Hrd1 closed the channel. Overall, this thesis provides strong evidence that
Hrd1 forms a protein-conducting channel in the ER membrane. A model is proposed,
whereby an affinity gradient between luminal and cytoplasmic binding sites provides
the driving force during retrotranslocation. | de |
| dc.contributor.coReferee | Steinem, Claudia Prof. Dr. | |
| dc.contributor.thirdReferee | Meinecke, Michael Prof. Dr. | |
| dc.contributor.thirdReferee | Faesen, Alexis Caspar Dr. | |
| dc.contributor.thirdReferee | Urlaub, Henning Prof. Dr. | |
| dc.subject.eng | Protein quality control | de |
| dc.subject.eng | Endoplasmic reticulum | de |
| dc.subject.eng | Ubiquitination | de |
| dc.subject.eng | Protein translocation | de |
| dc.subject.eng | ERAD | de |
| dc.subject.eng | Membrane proteins | de |
| dc.subject.eng | Protein folding | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-135C-F-7 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2020-06-25 | |
| dc.identifier.ppn | 1692333666 | |