We thank Dr. Yuval Reiss and Dr. William Breuer for critically reviewing the manuscript and Omri Alfassy for helping in the development of the Ura3-GFP screen assay. We also thank Dr. M. Hochstrasser and Dr. R. Kulka for plasmids and strains. This work was funded by the Israeli Academy of Sciences (grant 786/08) and by the United States-Israel Binational Scientific foundation (grant 2011253).

1. Zellkultur <ol><li> Transformation der geeigneten auxotrophen Hefe-Zellen, wie Try467 (Tabelle 2), die mit einem Plasmid, enthaltend (a) ein URA3 -degron Fusion und (b) einer zusätzlichen Stoffwechselmarker für Plasmidselektion und Wartung. HINWEIS: Ein Beispiel für ein geeignetes Plasmid ist YDpK-MET25p-deg1-FLAG-Vma12-Ura3 (LYS2) (Deg1- UV) Dies ist eine Hefe integrative Plasmid ein Fusionsprotein bestehend aus deg1 enthält - eine Degron aus der Hefe-Transkriptions abgeleitet. Faktor Mat2 17, FLAG-Epitop - zur Detektion durch Immunoblotting Vma12 - einem stabilen ER-Protein und Ura3 15,18 (Abbildung 3A). (Siehe Tabelle 3 für die Plasmide in dieser Studie und zusätzliche Beispiele für geeignete Plasmide verwendet.) </li><li> Für Plasmid Auswahl und Wartung, halten Zellen in Agar-Platten unter einem geeigneten Kunst Defined (SD) Medium ohne Aminosäure Selektionsmarker <sbis&gt; 19 (beispielsweise deg1 -UV ausgewählt und unter SD-LYS selektiven Medium gehalten). </li><li> Wachsen Zellen O / N bei 30 ° C in einem geeigneten selektiven Medium für SD Plasmids, bis eine stationäre Phase erreicht ist. Wachsen die Zellen in Teströhrchen oder in einer Platte mit 96 Vertiefungen, abhängig von der Anzahl von Proben (Abschnitt 2) untersucht werden. </li></ol> 2. Zellpräparation für Analysis <ol><li> Wenn eine kleine Anzahl von Proben (N≤15) werden untersucht werden, verdünnt, die Zellen und Ersetzen des Mediums wie folgt: <ol><li> Verdünnte 50 ul Zellen aus einem O / N-Kultur mit 150 &amp; mgr; l SD-Medium pro Vertiefung in einer Platte mit 96 Vertiefungen (clear bottom). Beachten, die erste Probe bezeichnet als Leer enthält nur Medium. </li><li> Bestimmen Sie die OD 600 Werte der Proben in der 96-Well-Platte mit Hilfe eines Mikroplatten-Reader. </li><li> Berechnung des tatsächlichen OD 600 in jeder Vertiefung durch Multiplizieren der OD 600 Lesen durch den Verdünnungsfaktor (x4), von whICH den Wert der Blindwert subtrahiert. Normalisierung der resultierenden Werte durch Dividieren durch 0,55, um eine berechnete Pfadlänge von 1 cm nach dem Beer-Lambert Gesetz erhalten. Hinweis, dieser Wert konstant ist, wenn die Messung der OD 600 von 200 ul der Zellen in einem Standard-96-Well-Platte. </li><li> Berechnet das genaue Volumen zum Erhalten Hefezellen bei äquivalenten Menge bis zu einer OD 600 von 0,25 und sie auf ein Eppendorf-Röhrchen benötigt. </li><li> Spin-down der Zellen bei hoher Geschwindigkeit (12.000 xg) für 1 min. </li><li> Entfernen Sie den Überstand und die Zellen resuspendieren in 1 ml des entsprechenden Selektionsmedium (siehe Abschnitt 3.1), um OD erhalten 600 von 0,25. </li><li> Transfer 200 ul der verdünnten Stämme zu einer neuen Vertiefung in einer Platte mit 96 Vertiefungen. </li></ol></li><li> Wenn eine große Anzahl von Proben (N&gt; 15) getestet werden soll, verdünnt, die Zellen und das Medium ohne vorherige OD 600 Messung ersetzt, wie folgt: <ol><li> Transfer 10 ul stationären Zellengrown O / N in einer 96-Well-Platte in eine neue 96-Well-Platte mit 190 ul (Verdünnung 1:20) der geeigneten selektiven Medien (siehe Abschnitt 3.1). </li></ol></li></ol> 3. Wachstum Kinetics in Flüssigkultur unter selektiven Bedingungen (GIL) <ol><li> Verwenden Sie die folgenden Medien für Wachstum Messungen mit dem Ura3-Degron Reporter: <ol><li> Für positive Kontrollen von Hefe Wachstum unter Nicht restriktiven Bedingungen, die Verwendung Plasmid selektive SD-Medien (siehe Abschnitt 1.2). Wenn keine Plasmid-Selektion erforderlich ist, beispielsweise bei Verwendung eines integrativen Plasmid wie deg1 -UV, SD-Medium, das alle erforderlichen Aminosäuren (SD vollständig) können verwendet werden. </li><li> Für experimentelle Proben Mess Wachstum unter restriktiven Bedingungen, verwenden Sie SD-URA Medium. </li></ol></li><li> Stellen Sie einen Multimode-Mikroplatten-Reader zu:. Einer Inkubationstemperatur von 30 ° C, OD 600 Messintervallen von 15 min und einer Orbital und lineare Schütteln Zyklen von 1 min alle sieben min &lt;br /&gt; Hinweis: Diese Dual-Mode-Schütteln wurde optimiert, um eine homogene Verteilung der Zellen zu erhalten; jedoch können auch andere Modi Schütteln oder sogar kein Schütteln so gut funktionieren. </li><li> Inkubieren der Zellen bei 30 ° C in einem Mikroplattenleser O / N oder bis die Zellen die stationäre Phase (12-24 h) erreicht. </li><li> Exportieren Sie die Rohdaten in ein Tabellenkalkulationsdatei. </li><li> Bestimmen Sie das Wachstum der Hefe Kinetik mit MDTcalc, eine maßgeschneiderte Software, die die minimale Zeit berechnet (in h) für Zellen, ihre Populationsgröße verdoppeln (Minimal Verdopplungszeit (MDT); für Details siehe Abschnitt 4). </li></ol> 4. Berechnung des Minimal-Verdopplungszeit (MDT): Grundsätze der MDTcalc Algorithm HINWEIS: Grundsätze der MDTcalc Algorithmus unter Verwendung einer repräsentativen Auswahl von Try467 Hefezellen (Tabelle 2) in SD-Medium gezüchtet abgeschlossen, sind in Tabelle 1 und Figur 1 dargestellt, die eine Datentabellenkalkulation und Wachstumsgrund, respectiv. <ol><li> Subtrahieren OD 600 Blindwerte in die entsprechende Wanne erhalten wird, von jeder der in der Probenwerte erhalten. Teilen die resultierenden Differenzen von 0,55 (für 96-Well-Platten), um die Lichtpfadlänge zu korrigieren. Zeichnen Sie die endgültigen Werte (Tabelle 1, Trans.) Gegen die Zeit, um die tatsächliche Hefe Wachstumskurve (OD 600 / h) (1A) zu produzieren. </li><li> Log 2 (OD 600) für jeden der transformierten Werten zu berechnen, um die logarithmische Wachstumsphase in eine lineare Kurve (1B) zu konvertieren. </li><li> Berechnen der Steigung einer 10 Zeitpunkte Intervall (N = 10), beginnend bei der Zeit 0 und Bewegen von einem Zeitpunkt, zu einer Zeit, bis N &lt;10 (Tabelle 1, Slope; 1C). </li><li> Berechnung der inversen Wert jeder Steigung, die Zeit für die Zellpopulation erforderlich ist, um doppelt zu erhalten (Tabelle 1, 1 / Steigung; 1C). Berechnen Sie die minimale1 / Slope-Wert, um die MDT (; 1C Tabelle 1, schwarzes Rechteck) zu bestimmen. Entpacken Sie die berechnete MDT für Zellen in der Probe (Tabelle 1, 1 / Slope, schwarzes Rechteck) aus dem gewählten Zeitintervall (Tabelle 1, durch rotes Rechteck skizziert, 1B, 1D). HINWEIS: Die Geschwindigkeit des Zellwachstums auf SD-URA Medium ist umgekehrt proportional zu der MDT. </li></ol> 5. Mit MDTcalc <ol><li> Kopieren Sie die Programmdateien ergänzt in einem bestimmten Ordner auf dem Computer. </li><li> Stellen Sie sicher, das Tabellenkalkulationsdatei, die die Wachstumsdaten gespeichert und geschlossen. Öffnen Sie die MDTcalc Anwendungsprogramm, um den Startbildschirm erscheint sehen. <ol><li> Legen Sie die erforderlichen Informationen, wie in Abbildung 2 gezeigt: <ol><li> Unter &quot;Dateipfad&quot;, klicken Sie auf das Rechteck über das Recht auf die Kalkulationstabelle Datei zu suchen. </li><li> Unter &quot;Blattname&quot;, schreiben die Tabelle Namen, wo ter Rohdaten befindet. </li><li> Unter &#39;Startposition&#39;, legen Sie die Tabellenkalkulation Koordinate Zeitpunkt 0 der ersten Probe. </li><li> Unter &#39;Time Spalte &quot;, legen Sie die Buchstaben die Festlegung des Orts der Zeitpunkt Spalte (Zeit sollte in Sekunden zur Verfügung gestellt werden). </li><li> Unter &#39;Blank Spalte &quot;, legen Sie die Buchstaben definieren die Position der leeren Spalte (in der Regel, aber nicht notwendigerweise in A1 der 96-Well-Platte). </li><li> Unter &#39;OD-Wert &quot;, wählen Sie den minimalen transformiert OD 600-Wert für die Einleitung der MDT Berechnung (in der Regel 0,15-0,25) erforderlich. Wird dieser Wert verhindert Berechnungen der MDT für Kulturen in Lag-Phase wegen zu hoher Verdünnung, so dass MDT ist nur für Proben, die den definierten OD 600-Wert oder höher zu erreichen, berechnet. </li></ol></li><li> Sobald der letzte Wert eingegeben wurde, drücken Sie die &quot;berechnen&quot; -Button am unteren Rand des Bildschirms. Stellen Sie sicher, dass der Fortschrittsbalken auf der rechten Seite allmählich in gReen. Die Kalkulationstabellendatei während des Rechenprozesses nicht öffnen. </li><li> Exportieren der zwei Sätze von Daten, die automatisch auf dem Bildschirm bei der Beendigung der Berechnung MDT in ein Arbeitsblatt angezeigt. Stellen Sie sicher, dass die Daten enthält: Start (a) die MDT-Wert für jeden gut, dass die minimale OD-Wert-Test (wie in Abschnitt 5.2.1.6 festgelegt) und (b) den Anfangszeitpunkt des Intervalls aus dem die MDT berechnet gibt ( Zeit). </li></ol></li></ol>

<ol>
	<li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annual review of biochemistry. 67, 425-479 (1998).</li><li>Ravid, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+degradation+signals+in+the+ubiquitin-proteasome+system.">Diversity of degradation signals in the ubiquitin-proteasome system.</a> Nature reviews. Molecular cell biology. 9, 679-689 (2008).</li><li>Willems, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCF+ubiquitin+protein+ligases+and+phosphorylation-dependent+proteolysis.">SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.</a> Philos Trans R Soc Lond B Biol Sci. 354, 1533-1550 (1999).</li><li>Tyers, M., Jorgensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+and+the+cell+cycle:+with+this+RING+I+do+thee+destroy.">Proteolysis and the cell cycle: with this RING I do thee destroy.</a> Curr Opin Genet Dev. 10, 54-64 (2000).</li><li>Johnson, P. R., Swanson, R., Rakhilina, L., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signal+masking+by+heterodimerization+of+MAT&#945;2+and+MAT+a1+blocks+their+mutual+destruction+by+the+ubiquitin-proteasome+pathway.">Degradation signal masking by heterodimerization of MAT&#945;2 and MAT a1 blocks their mutual destruction by the ubiquitin-proteasome pathway.</a> Cell. 94, 217-227 (1998).</li><li>Furth, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum/nuclear+envelope.">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Molecular Biology of the Cell. 22, 4726-4739 (2011).</li><li>Fredrickson, E. K., Gallagher, P. S., Clowes Candadai, S. V., Gardner, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+recognition+in+nuclear+protein+quality+control+degradation+is+governed+by+exposed+hydrophobicity+that+correlates+with+aggregation+and+insolubility.">Substrate recognition in nuclear protein quality control degradation is governed by exposed hydrophobicity that correlates with aggregation and insolubility.</a> J Biol Chem. 10, (2013).</li><li>Rosenbaum, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorder+Targets+Misorder+in+Nuclear+Quality+Control+A+Disordered+Ubiquitin+Ligase+Directly+Recognizes+Its+Misfolded+Substrates.">Disorder Targets Misorder in Nuclear Quality Control A Disordered Ubiquitin Ligase Directly Recognizes Its Misfolded Substrates.</a> Molecular cell. 41, 93-106 (2011).</li><li>Ciechanover, A., Hod, Y., Hershko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heat-stable+polypeptide+component+of+an+ATP-dependent+proteolytic+system+from+reticulocytes.">A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.</a> Biochemica., &amp; Biophysical Research Communications. 81, 1100-1105 (1978).</li><li>Finley, D., Ulrich, H. D., Sommer, T., Kaiser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+system+of+Saccharomyces+cerevisiae.">The ubiquitin-proteasome system of Saccharomyces cerevisiae.</a> Genetics. 192, 319-360 (2012).</li><li>Klis, F. M., Boorsma, A., De Groot, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+construction+in+Saccharomyces+cerevisiae.">Cell wall construction in Saccharomyces cerevisiae.</a> Yeast. 23, 185-202 (2006).</li><li>Zhou, W., Ryan, J. J., Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analyses+of+sumoylated+proteins+in+Saccharomyces+cerevisiae.+Induction+of+protein+sumoylation+by+cellular+stresses.">Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.</a> J Biol Chem. 279, 32262-32268 (2004).</li><li>Alani, E., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+type+of+fusion+analysis+applicable+to+many+organisms:+protein+fusions+to+the+URA3+gene+of+yeast.">A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.</a> Genetics. 117, 5-12 (1987).</li><li>Swanson, R., Locher, M., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+ubiquitin+ligase+of+the+nuclear+envelope/endoplasmic+reticulum+that+functions+in+both+ER-associated+and+Matalpha2+repressor+degradation.">A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.</a> Genes Dev. 15, 2660-2674 (2001).</li><li>Ravid, T., Kreft, S. G., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+soluble+substrates+of+the+Doa10+ubiquitin+ligase+are+degraded+by+distinct+pathways.">Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways.</a> EMBO J. 25, 533-543 (2006).</li><li>Gilon, T., Chomsky, O., Kulka, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signals+for+ubiquitin+system+proteolysis+in+Saccharomyces+cerevisiae.">Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.</a> Embo J. 17, 2759-2766 (1998).</li><li>Hochstrasser, M., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+degradation+of+a+transcriptional+regulator:+the+yeast+2+repressor.">In vivo degradation of a transcriptional regulator: the yeast 2 repressor.</a> Cell. 61, 697-708 (1990).</li><li>Metzger, M. B., Maurer, M. J., Dancy, B. M., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+a+cytosolic+protein+requires+endoplasmic+reticulum-associated+degradation+machinery.">Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery.</a> J Biol Chem. 283, 32302-32316 (2008).</li><li>Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Yeast Genetics and Molecular Biology. 194, (1991).</li><li>Carvalho, P., Goder, V., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ubiquitin-ligase+complexes+define+convergent+pathways+for+the+degradation+of+ER+proteins.">Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.</a> Cell. 126, 361-373 (2006).</li><li>Denic, V., Quan, E. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luminal+surveillance+complex+that+selects+misfolded+glycoproteins+for+ER-associated+degradation.">A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.</a> Cell. 126, 349-359 (2006).</li><li>Wu, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+catalytic+residue+in+the+ubiquitin-conjugating+enzyme+family.">A conserved catalytic residue in the ubiquitin-conjugating enzyme family.</a> Embo J. 22, 5241-5250 (2003).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Fluoroorotic+acid+as+a+selective+agent+in+yeast+molecular+genetics.">5-Fluoroorotic acid as a selective agent in yeast molecular genetics.</a> Methods Enzymol. 154, 164-175 (1987).</li><li>Alfassy, O. S., Cohen, I., Reiss, Y., Tirosh, B., Ravid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placing+a+disrupted+degradation+motif+at+the+C+terminus+of+proteasome+substrates+attenuates+degradation+without+impairing+ubiquitylation.">Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.</a> J Biol Chem. 288, 12645-12653 (2013).</li><li>Cronin, S. R., Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+degradation+with+green+fluorescent+protein.">Measuring protein degradation with green fluorescent protein.</a> Methods in Enzymology. 302, 58-73 (1999).</li></ol>

The authors have nothing to disclose.

Hier beschreiben wir einen Test auf Basis von Zellwachstum zur Bestimmung der relativen Proteinabbauraten, genannt &#39;Wachstumskinetik in Flüssigkultur unter selektiven Bedingungen &quot;(GIL). Die Gils Assay hat mehrere Vorteile: Es ist einfach einzurichten, Datenerfassung und Analyse ist einfach und es ist extrem modular ist. Folglich GILS können gleichzeitig mehrere Proben in einer benutzerfreundlichen Multi-Well-Plattenformat, die angepasst werden können, um für Anwendungen mit hohem Durchsatz Automatisierung ...

Das Ubiquitin-Proteasom-Abbausystem ist ein Hauptregulierungs Maschine, die bei der Aufrechterhaltung der Homöostase Protein in Eukaryoten beteiligt ist. Der UPS-Konjugate zunächst mehrere Ubiquitin-Moleküle an ein Zielprotein nach der die Poly-Ubiquitin-markierten Protein durch das 26S Proteasom abgebaut. In den meisten Fällen ist der geschwindigkeitsbestimmende Schritt für die Ubiquitin-vermittelten Abbau Substrat Ubiquitinierung von E2 konjugierenden Enzyme und Liga E3-Enzymen (E3 Ligasen) 1. Folglich intrazelluläre Stabilität spezifischer Proteine ​​spiegelt ihre Anfälligkeit für Ubiquitin-Konjugation und die Aktivität ihres zugehörigen Ubiquitinierung Enzyme. E3-Ligasen sind die Hauptsubstraterkennung Komponenten des UPS. Als solche sind diese Enzyme erkennen degrons innerhalb ihrer Substrate, die entweder nicht vorhanden oder nicht in ihrer stabilen Gegen 2 ausgesetzt sind. Zum Beispiel sind viele Regulatoren des Zellzyklus must synthetisiert und in einer zeitlich bestimmten Weise, um die Zellzyklusprogression in Ordnung zu halten abgebaut werden. Der Abbau dieser Proteine ​​wird häufig durch Phosphorylierung gesteuert durch zelluläre Signale regulierte Kinasen 3,4 vermittelt. Auf der anderen Seite, sind aberrant-gefaltete Proteine ​​durch kryptische degrons anerkannt. Dies sind Bereiche, die normalerweise in der nativen Struktur verborgen sind und werden beim Aufbau Störung ausgesetzt. Solche degrons gehören hydrophoben Domänen 5-7 und intrinsisch ungeordneten Segmente 8. Da der Samen Entdeckung des Ubiquitin-Proteinabbau und Charakterisierung der Grundlagen in Retikulozyten-Lysaten 9 war Hefegenetik an der Entdeckung von vielen der Komponenten des Ubiquitin-Systems 10. Der Erfolg von Hefe als Modellorganismus für die systematische Analyse von Proteinabbau durch die USV ist vor allem aufgrund der Tatsache, dass die USV ist hochly konserviert in allen Eukaryoten 4, verbunden mit ihrer Zugänglichkeit als Versuchssystem. Tatsächlich werden auf Hefe basierenden Systemen üblicherweise verwendet, um die Wirkmechanismen der Ubiquitinierung Maschinen entziffern. Studieren Proteinabbau durch biochemische Mittel erfordert in der Regel Herstellung von Zellextrakten. Während tierische Zellproteinen kann unter relativ milden Bedingungen, die Proteinwechselwirkungen und Funktion zu erhalten extrahiert werden, das Vorhandensein eines robusten Zellwand der Hefe 11 benötigt deutlich härteren Bedingungen, die Störung der Proteingewinnung beeinflussen können. Tatsächlich unterschiedliche Verfahren für Hefe Zellaufschluss variieren erheblich in ihrer Fähigkeit, intakte Proteine ​​in Mengen, die ihre relative Zellüberfluss korrekt darstellen zu erholen. Weitere Ungenauigkeit ist inhärent in den verschiedenen zur Bestimmung Abbauraten von spezifischen Proteinen angewandten Methoden: Metabolische Markierung basierten &#39;Pulse-Chase-Experimente, gefolgt von immunoprecipitation, um spezifische Proteine ​​12 zu isolieren, ist häufig nicht streng quantitativ. Somit wird, wenn Proteinabbau wird durch dieses Verfahren im Vergleich, extra Vorsicht bei der Interpretation der Ergebnisse geboten. Um diesen Nachteil zu umgehen, kann eine alternative Cycloheximid (CHX) chase Assay verwendet werden 12. In diesem Assay wird die Übersetzung Inhibitor-Zellkulturen und zeitlichen Veränderungen der Protein stationären Ebenen hinzugefügt werden anschließend überwacht. Dennoch ist die Verwendung von CHX Proteine ​​mit relativ kurzen Halbwertszeiten (&lt;90 min), als langfristige Hemmung der Proteinsynthese ist zytotoxisch beschränkt. Bemerkenswert ist, beide der oben genannten Tests erfordern die Verwendung von Protein-spezifischen Antikörpern, die nicht immer verfügbar sind. Um diese technischen Einschränkungen zu überwinden, haben die Forscher mehrere Ansätze, die nicht Zellextraktion und direkte Protein Handhabung erfordern entwickelt. Ein Ansatz basiert auf der Einrichtung von auxotrophen yea basiertst Stämme, durch die Deletion von Genen wesentlichen metabolischen Enzyme codieren, erhalten werden. Solche Gene umfassen HIS3, LEU2, LYS2 und TRP1, kodierend für Enzyme, die für die Aminosäure-Biosynthese erforderlich ist, als auch URA3, die OMP-Decarboxylase codiert (Ura3), ein essentielles Enzym der Pyrimidin-Biosynthese Ribonukleotid. Ura3 wurde vielfach in der Proteinabbaustudien eingesetzt. In diesen Assays rettet konstitutive Expression Ura3 Wachstum von Zellen in ura3 Uracil-Mangelmedium 13. Folglich Destabilisierung Ura3 durch die Fusion einer Degron kann das Zellwachstum auf Minimalmedium ohne Uracil verringern. Diese Methode wurde in verschiedenen Proteinabbaustudien eingesetzt, einschließlich der Festlegung von Abbaufaktoren 5, E3 Ligasen 14 und Hilfs Ubiquitinierung Faktoren 15 und die Entdeckung von neuen USV degrons 16. Alle diese Verfahren verwendet das Zellwachstum auf Agarplatten als Testanzeige. Jedoch ter Wachstumskriterium (positive / negative Wachstum), während robuste und effiziente, ist meist qualitative und nicht quantitative Informationen, die wichtig für die Bewertung Potenz eines Degron oder den relativen Beitrag der verschiedenen Hilfsabbaufaktoren ist. Wir haben daher entwickelt und verwendet Hefevektoren und Screeningverfahren zur systematischen und quantitativen Analyse der Proteinabbau durch das URA3 -degron Fusionssystem. Das Protokoll basiert auf einer einfach zu handhabenden Test, der Wachstumskinetik misst in Flüssigkultur unter selektiven Bedingungen (GIL) und auf der Erzeugung von Standardwachstumskurven. Lag die exponentielle (log) und der stationären Phase - Hefe Wachstumskinetik werden durch drei Hauptphasen gekennzeichnet. Berechnung der Hefe-Replikations Kinetik während der log-Phase unter selektiven Bedingungen, die durch die Höhe der Expression des URA3-Degron bestimmt wird, liefert eine objektive quantitative Messung of Proteinabbau. Dieses Verfahren kann zum Messen und Vergleichen Abbauraten von mehreren UPS Substrate gleichzeitig in mehrere Stämme und unter verschiedenen Bedingungen verwendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Name of Material/ Equipment</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:184px;">Difco yeast nitrogen base w/o amino acids and ammonium sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td rowspan="6" style="width:339px;">For yeast growth on SD minimal media.&#160; Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium sulfate</td>
			<td>Sigma - Aldrich</td>
			<td>A4418</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma - Aldrich</td>
			<td>16325</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenine</td>
			<td>Sigma - Aldrich</td>
			<td>A8626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uracil</td>
			<td>Sigma - Aldrich</td>
			<td>U0750</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amino acids</td>
			<td>Highest purity available&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">96 well plates</td>
			<td style="width:149px;">Nunc</td>
			<td style="width:119px;">167008</td>
			<td>Any other compatible brand can be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Cyclohexamide&#160;</td>
			<td style="width:149px;">Sigma - Aldrich</td>
			<td style="width:119px;">C7698</td>
			<td>Working conc. 0.5 mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Infinite 200 PRO series</td>
			<td style="width:149px;">Tecan</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">For yeast incubation and OD 600 measurements. Any other compatible temp-controled reader can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">MDTcalc</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td>Experimental software</td>
		</tr>
	</tbody>
</table>

reporter-based growth assay for systematic analysis of protein degradation

Der Proteinabbau durch das Ubiquitin-Proteasom-System (UPS) ist ein wichtiger Regulationsmechanismus für Proteinhomöostase in allen Eukaryonten. Die Standardmethode zur Bestimmung der intrazellulären Proteinabbaus beruht auf biochemischen Assays für folgende die Kinetik der Proteinniedergang. Solche Verfahren sind oft umständlich und zeitaufwendig und daher nicht zugänglich sind Versuche zur Beurteilung mehrere Substrate und Abbaubedingungen ab. Als Alternative wurden Zellwachstum basierenden Assays entwickelt worden, die sich in ihrer herkömmlichen Format Endpunktassays, die nicht quantitativ relativen Veränderungen in der Proteinspiegel zu bestimmen können. Hier beschreiben wir eine Methode, die originalgetreu bestimmt Veränderungen in der Proteinabbauraten durch Kopplung an Hefezelle-Wachstumskinetik. Das Verfahren basiert auf einem System, in dem festgelegten Auswahl Uracil-Auxotrophie des URA3 -deleted Hefezellen durch eine exogen exprimierten Reporterproteins gerettet basierend, Einer Fusion zwischen dem wesentlichen URA3-Gen und einem Abbau Determinante (Degron) besteht. Das Reporterprotein ist so ausgelegt, dass ihre Syntheserate konstant ist, während seine Abbaurate wird durch die Degron bestimmt. Zellwachstum in Uracil-defizientem Medium proportional zu der relativen Pegel Ura3 sind Wachstumskinetik völlig abhängig von der Reporter-Protein-Abbau. Diese Methode misst genau Veränderungen der intrazellulären Proteinabbau Kinetik. (A) Die Beurteilung der relative Beitrag der bekannten Ubiquitinkonjugierenden Faktoren Proteolyse (b) E2 konjugierende Enzym Struktur-Funktions-Analysen (c) Identifizierung und Charakterisierung von neuen degrons: Es wurde angewandt. Anwendung des degron- URA3 -basierten System überwindet die Abbaufeld Protein, wie es auch zur Überwachung von Veränderungen der Proteinspiegel mit Funktionen der anderen zellulären Wege angepasst zu werden.

Untersuchung der Rolle der Enzyme der Doa10 Weg zum Abbau eines Reportersubstrat Um die Gültigkeit der GIL Verfahren wurde mit einem herkömmlichen Assay Abbau verglichen zu testen. Dieses Experiment beurteilt die relativen Beitrag der Komponenten der ER-Membran lokalisiert Doa10 E3-Ligase-Komplex 20,21 zum Abbau des Proteins Qualitätskontrolle Reportersubstrat Deg1- VU (3A). Die Deg1- VU Plasmid wurde in Wildtyp-Zellen</em...

Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Reporter-basierten Wachstumstest für die systematische Analyse von Proteinabbau

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

reporter-based-growth-assay-for-systematic-analysis-protein

Research

JoVE Journal

Biology

10.6K Aufrufe.  The Hebrew University of Jerusalem. Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Serie: Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Aktin Co-Sedimentation Assay; für die Analyse von Protein-Bindung an F-Aktin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Forschung

Biologie

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Growth-basierte Bestimmung und biochemische Bestätigung der genetischen Voraussetzungen für Proteinabbau in<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cycloheximid Chase-Analyse von Proteinabbau in<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Assays für den Abbau fehlgefalteter Proteine ​​in Zellen

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

Ein Hydroponisches Co-Kultivierungssystem zur gleichzeitigen und systematischen Analyse von Pflanzen- / Mikroben-Molecular-Wechselwirkungen und Signalgebung

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Präzise, Hochdurchsatz-Analyse des Bakterienwachstums

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase Komplementierung Imaging Assay in Nicotiana Benthamiana Blätter für Transient Bestimmung Proteinprotein Interaktion Dynamik

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...