Die HECT-Ligase Hul5, eine neue Komponente der ER-assoziierten Proteindegradation

Abstract

Die meisten sekretorischen Proteine der eukaryontischen Zellen erreichen durch das endoplasmatische Retikulum (ER) den sekretorischen Signalweg. Sie gelangen vom Zytoplasma durch einen Kanal in der ER-Membran in das ER, wo sie ihre native Konformation erhalten. Das ER enthält ein strenges Qualitätskontrollsystem, welches fehlgefaltete Proteine erkennt, im ER zurückhält und letztendlich der ER-assoziierten Degradation (ERAD) zuführt. Die ER-Qualitätskontrolle und die ER-assoziierte Degradation sind eng miteinander verknüpft, und werden unter der Bezeichnung ER-Qualitätskontrolle und assoziierte Degradation (ERQD) zusammengefasst. Die ERQD ist von der Hefe bis hin zum Menschen ein hoch konservierter Prozess. Aus diesem Grund wird die Hefe Saccharomyces cerevisiae als Modellorganismus zur Erforschung solcher sogenannter „housekeeping“ Prozesse genutzt.

In dieser Arbeit wurden durch einen genomweiten Screen neue Komponenten der ER-assoziierten Degradation identifiziert. Für diesen Screen wurden die EUROSCARF Hefe-Deletionsbank und das Screeningsubstrat Sec61-2L verwendet. Die Deletionsbank besteht aus etwa 5000 diploiden S. cerevisiae Stämmen mit je einer homozygoten Einfachdeletion. Bei dem Screeningsubstrat Sec61-2L handelt es sich um das nicht glykosylierte, mutierte Translokonprotein Sec61-2 und einer C-terminalen zytosolischen Fusion mit der 3-Isopropylmalat-Dehydrogenase (Leu2). Das für Sec61-2L kodierende Plasmid wurde in die leu2-auxotrophen Deletionsstämme transformiert und der Wachstumsphänotyp bei 38° auf Leucin-defizientem Medium getestet. Aufgrund der Punktmutation faltet sich Sec61-2 bei 38°C in einer Art und Weise, dass es der ER-Degradation unterliegt. Nur wenn Sec61-2L stabil vorliegt, also in Deletionsstämmen mit einem Defekt in der Erkennung oder der Degradation des fehlgefalteten Sec61-2L, ist ein Wachstum auf Leucin-defizientem Medium möglich. Auf diese Weise konnten über 40 bisher unbekannte, potentielle Komponenten der ER-Qualitätskontrolle und ER-assoziierten Degradation identifiziert werden. Unter anderem wurde durch diesen genomischen Screen die E4-Ligase Hul5 als Komponente des ERAD für dieses nicht glykosylierte Substrat gefunden. Des Weiteren war bereits durch einen entsprechenden Screen mit dem Substrat CTL* bekannt, dass Hul5 auch am Abbau dieses glykosylierten ERAD-Substrats beteiligt ist.

In der vorliegenden Arbeit wurde nachgewiesen, dass für den vollständigen Abbau der ERAD-Substrate Sec61-2Lmyc und CTL*myc die katalytische Funktion der E4-Ligase Hul5 benötigt wird. Außerdem wurde gezeigt, dass der Abbau der Substrate Sec61-2Lmyc und CTL*myc im Wildtypstamm sowie in der HUL5 Deletionsmutante am N-Terminus einsetzt und über definierte Zwischenprodukte verläuft. Im Wildtypstamm kann auf diese Weise der Abbau der Substrate vollständig verlaufen. Im Gegensatz dazu erfolgt in der HUL5 Deletionsmutante ein Abbruch der Degradation am Proteasom, was zu einer Akkumulation der C-terminalen Abbaufragmente truncSec61-2Lmyc und truncCTL*myc führt.

Des Weiteren wurde gezeigt, dass für den Abbau des N-terminalen Anteils von CTL*myc keine Extraktion des Proteins aus der ER-Membran notwendig ist. Demzufolge muß der N-Terminus von CTL*myc durch die ER-Membran in das Zytosol der Zelle ragen, wo die Ubiquitinierung und die Degradation des Substrats einsetzen. Außerdem wurde gefunden, dass auch das Proteasom an der Extraktion von CTL*myc aus dem ER beteiligt ist.

Es ist bekannt, dass die E4-Ligase Hul5 gemeinsam mit dem deubiquitinierenden Enzym Ubp6 und dem Ubiquitin-konjugierenden Enzym Ubc4 den Abbau anderer proteasomaler Substrate regulieren kann. In dieser Arbeit wurde gezeigt, dass die Degradation des ERAD-Substrats CTL*myc durch Hul5, jedoch nicht durch Ubp6 und Ubc4 beeinflusst wird. Dieses Ergebnis gibt Hinweise auf weitere mit Hul5 agierende deubiquitinierende und Ubiquitin-konjugierende Enzyme.

The endoplasmic reticulum (ER) plays an essential role in the folding and maturation of newly synthesized proteins in the secretory pathway. It provides an enviroment optimized for folding, oxidation and oligomeric assembly of proteins translocated into the ER membrane. Folding in the ER is assisted by a large variety of folding enzymes, molecular chaperones and folding sensors. To ensure the fidelity of the maturation process, exit from the ER is regulated by a stringent quality control system that inhibits the secretion of incompletely folded or misfolded proteins. These misfolded polypeptides and orphan subunits are subsequently subjected to ER-associated degradation (ERAD). The ERAD process requires retrotranslocation of the misfolded proteins across the ER membrane into the cytoplasm and subsequent degradation by the ubiquitin-proteasome pathway.

Degradation of a protein by the ubiquitin-proteasome pathway involves two discrete and successive steps: the tagging of the substrate by covalent attachment of multiple ubiquitin molecules and the degradation of the tagged protein by the 26S proteasome complex. Conjugation of ubiquitin, a highly evolutionarily conserved 76-residue polypeptide, to the protein substrate proceeds via a three-step cascade mechanism. Initially, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction to generate a high-energy thiol ester intermediate, E1-S?ubiquitin. One of several E2 enzymes (ubiquitin-conjugating enzymes, UBCs) transfers the activated ubiquitin moiety from the E1, via an additional high energy thiol ester intermediate, E2-S?ubiquitin, to the substrate that is specifically bound to a member of the ubiquitin-protein ligase family, E3. The ubiquitin molecule is generally transferred to an ?-NH2 group of an internal Lys residue in the substrate to generate a covalent isopeptide bond. By successively adding activated ubiquitin moieties to internal Lys residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized. In some cases, multiubiquitination requires the additional activity of certain ubiquitin-chain elongation factors, the so-called E4-ligases. The polyubiquitin chain is recognized by the downstream 26S proteasome complex. The proteasome is a large, 26S, multicatalytic protease. It is composed of two subcomplexes: a 20S core particle (CP) that carries the catalytic activity and a 19S regulatoy particle (RP). The regulatory particle recognizes the substrate-bound ubiquitin chain, unfolds the substrate and directs its translocation into the core particle. Critical components of the regulatory particle include ubiquitin receptors, deubiquitining enzymes, and six ATPases that have been implicated in substrate unfolding and translocation. The 19S regulatory particle allows the entry of the substrate into the proteolytic chamber of the 20S core particle. The substrate is degraded by the multicatalytic proteases of the 20S core particle, short peptides derived from the substrate are relased.

It is important to note that the accumulation of misfolded proteins in the ER, especially under conditions of stress, triggers activation of a wide range of genes encoding proteins of the secretory pathway. This is the so-called unfolded protein response (UPR). Several of the UPR-induced proteins are involved in protein folding and glycosylation in the ER and in ER-associated degradation. Thus, quality control in ER and ERAD, abbreviated as ERQD (ER quality control and associated degradation) and UPR are tightly coordinated processes.

Because ERQD is highly conserved from yeast to man, the easy amenability of yeast to genetic and molecular biological studies combined with the knowledge of its genome and proteome makes it a preferred organism to study such "houskeeping" functions of eukaryotic cells. To gain deeper insight into the molecular mechanism of ER quality control and associated degradation a genome-wide screen was performed, using the EUROSCARF yeast library, consisting of about 5000 Saccharomyces cerevisiae strains, each deleted for a single non-essential gene. The use of this library for screening mutants of ERAD is possible, because cells tolerate a defect in the process as long as the unfolded protein response is intact. A chimeric protein (Sec61-2L) consisting the nonglycosylated ER-membrane-located ERQD substrate Sec61-2 fused to cytoplasmic 3-isopropylmalate dehydrogenase, the Leu2 protein, was used as a substrate for the genome wide screen. Cells carrying a LEU2 deletion can only grow on media lacking leucine when the chimeric protein Sec61-2L is not degraded. Thus, only mutant cells defective in an ERQD component can grow. The generated screen yielded about 40 mutants exhibiting a reproducible growth phenotype. Among these, a strong complemetation in the strain carrying the ?hul5 deletion was found.

Hul5 is known to act as a E4-ligase being specialized in the elongation of ubiquitin chains. Whether Hul5 is also able to promote the initial recognition of a substrate by the proteasome is still unknown. At the beginning of this work an interaction of Hul5 with Rpn2, a non-ATPase subunit of the proteasome which is located in the base of the regulatory particle, was established. It was shown, that the enzymatic activity of Hul5 is stimulated by its interaction with the proteasome. The chain-elongating activity of Hul5 functions specifically in opposition to the deubiquitinating activity of Ubp6, a deubiquitinating enzyme (DUB) interacting with the Rpn1 subunit of the 19S cap of the proteasome.

To test biochemically whether Hul5 is a component of the ERAD machinery, the degradation of the substrates CTL* and Sec61-2L was analysed by cycloheximide decay and pulse-chase analyses. Degradation kinetics of CTL* with a CPY specific antibody did not show any evidence for an involvement of Hul5 in ER-associated degradation. However, a C-terminally myc-tagged version of the substrates and the use of an myc specific antibody provided finally insight into the degradation of CTL*myc and Sec61-2Lmyc in the ?hul5 mutant anticipated from the genetic screen: It was able to show, that the ERAD substrates CTL*myc and Sec61-2L are degraded in the wildtype as in the HUL5 deletion strain in a stepwise manner starting at the N-terminus. In wild type cells the substrates are degraded completely, whereas in the ?hul5 mutant truncated forms of CTL*myc and Sec61-2L, truncCTL*myc and truncSec61-2Lmyc are accumulated. The degradation of the N-terminal part of CTL*myc and Sec61-2Lmyc proceeds in the ?hul5 mutant. However, the degradation of the C-terminal part of the substrates requires the E4-ligase Hul5. The degradation of the substrates CTL*myc and Sec61-2Lmyc is halted in the ?hul5 mutants. It is shown, that the interruption of the degradation depends on the missing chain-elongating activity of Hul5. The fragments truncCTL*myc and truncSec61-2Lmyc consist of the cytosolic Leu2myc-domain and a part of the CPY* or the Sec61-2L domain. Interestingly, the molecular mass of the fragments truncCTL*myc and truncSec61-2Lmyc according to their behaviour in SDS-PAGE seems to be the same. This astonishs as CTL*myc and Sec61-2L are completely different substrates, only the cytosolic Leu2myc-domain is common on both substrates. For other ERAD-substrates like Sec61-2, CPY*, CT* and CTG* the activity of Hul5 is not required for degradation.

The localization of truncCTL*myc gave a hint, how the degradation of the substrate CTL*myc is acchieved.The truncCTL*myc was partly found in the membrane and in the cytosol of the cell. Obviously, the degradation of the substrates can start, while they are located in the membrane. There is no previous extraction of the substrate from the ER membrane required for the start of degradation. The N-terminus of CTL*myc must loop out of the ER lumen, through the ER membrane into the cytosol of the cell, while the rest of the substrate is remaining in the membrane. The N-terminus of CTL*myc loops into the cytosol and is ubiquitinated and degraded by the proteasome. The experiments give evidence that the proteasome is involved in the extraction of the substrate CTL*myc out of the ER.

It is known, that Hul5 regulates the proteasomal degradation of special substrates together with the deubiquitinating Enzyme Ubp6 and the ubiquitin-conjugating enzyme Ubc4. However, the complete degradation of the substrate CTL*myc requires the activity of the E4-ligase Hul5, but the activity of Ubp6 and Ubc4 is not required.