Protein Parçalanabilirliklerinin Sistematik Analizi muhabiri tabanlı Büyüme Deneyi

Summary

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.

Abstract

Ubikitin-proteazom sistemi (UPS) tarafından protein bozunma bütün ökaryot protein homeostazı için önemli bir düzenleyici mekanizmadır. Hücre içi protein parçalanmasını belirleme standart yaklaşım protein düşüş kinetiğe için biyokimyasal deneyleri dayanır. Bu yöntemler, genellikle, zahmetli ve zaman alıcı ve birden çok alt-tabakalar ve ayrıştırma koşulları değerlendirme amaçlı deneyler bu nedenle uygun değildir. Bir alternatif olarak, hücre büyümesi dayalı tahliller kantitatif protein seviyelerindeki azalma şeklinde tespit edemez bunlar genellikle geleneksel biçimi, son-nokta deneylerde, olduğu, geliştirilmiştir.

Burada sadakatle maya hücresi büyüme kinetikleri olarak bağlanarak protein yıkımı oranlarındaki değişiklikleri belirleyen bir yöntem açıklanmaktadır. Yöntem URA3 ürasil oksotrofi maya hücreleri bir dışsal olarak ifade raportör protein tarafından kurtarıldı -silinmesi kurulmuş bir seçim sistemine dayanır, Temel URA3 geni ve bozunma belirleyici (degron) arasında bir füzyon oluşturulmuştur. Raportör proteini, bozulma oranı degron belirlenir kaydıyla, sentez oranının sabit olacağı şekilde tasarlanmıştır. Urasil-eksikli ortam hücre büyümesi URA3 nispi seviyelerine orantılı olduğu için, büyüme kinetikleri, raportör protein degradasyonu tamamen bağlıdır.

Bu yöntem doğru bir hücre içi protein yıkımı kinetik değişiklikleri ölçer. (A) E2 bağlayıcı enzim yapı-fonksiyon (c) tanımlanması ve yeni degrons karakterizasyonu analizleri proteoliz (b) bilinen ubikuitinden conjugating faktörlerin göreceli katkısını Değerlendirme: Bu uygulanmıştır. Aynı zamanda diğer hücresel yolların fonksiyonlar ile ilgili bir protein seviyelerinin izlenmesini değişikliklere adapte edilebilir degron- URA3 merkezli bir sistemin uygulanması, protein degradasyon alanını aşar.

Introduction

The ubiquitin-proteasome degradation system is a major regulatory machine, which has been implicated in the maintenance of protein homeostasis in all eukaryotes. The UPS initially conjugates multiple ubiquitin molecules to a target protein after which the poly-ubiquitin-tagged protein is degraded by the 26S proteasome. In most cases, the rate limiting step for ubiquitin mediated degradation is substrate ubiquitylation, mediated by E2 conjugating enzymes and E3 ligating enzymes (E3 Ligases)¹. Consequently, intracellular stability of specific proteins reflects their susceptibility to ubiquitin-conjugation and the activity of their cognate ubiquitylation enzymes.

E3 ligases are the principal substrate recognition components of the UPS. As such, these enzymes recognize degrons within their substrates that are either absent or not exposed in their stable counterparts ². For example, many regulators of the cell cycle must be synthesized and degraded in a temporally specific manner in order to keep cell cycle progression in order. The degradation of these proteins is often controlled by phosphorylation, mediated by cell-signaling regulated kinases ^3,4. On the other hand, aberrantly-folded proteins are recognized through cryptic degrons. These are regions that are normally hidden in the native structure and are exposed upon structure perturbation. Such degrons include hydrophobic domains ^5-7 and intrinsically disordered segments⁸. 

Since the seminal discovery of the ubiquitin-system for protein degradation and characterization of its fundamentals in reticulocyte lysates⁹, yeast genetics was instrumental in discovering many of the components of the ubiquitin system¹⁰. The success of yeast as a model organism for systematic analysis of protein degradation by the UPS is mainly due to the fact that the UPS is highly conserved in all eukaryotes⁴, coupled with their amenability as an experimental system. Indeed, yeast-based systems are commonly employed to decipher the mechanisms of action of the ubiquitylation machinery.

Studying protein degradation by biochemical means usually requires preparation of cell extracts. While animal cell proteins can be extracted under relatively mild conditions that preserve protein interactions and function, the presence of a robust cell wall in yeast¹¹ requires considerably harsher disruption conditions which may affect protein recovery. Indeed, different procedures for yeast cell disruption vary considerably in their capacity to recover intact proteins in amounts that correctly represent their relative cellular abundance. Further inaccuracy is inherent in the different methods employed for determining degradation rates of specific proteins: Metabolic labeling-based 'pulse-chase' experiments followed by immunoprecipitation, to isolate specific proteins¹², is often not strictly quantitative. Thus, when protein degradation is compared by this method, extra caution should be exercised in interpreting the results. To circumvent this drawback, an alternative cycloheximide (CHX) chase assay can be employed¹². In this assay, the translation inhibitor is added to cell cultures and temporal changes in protein steady-state levels are subsequently monitored. Nevertheless, the usage of CHX is limited to proteins with relatively short half-lives (< 90 min), as long-term inhibition of protein synthesis is cytotoxic. Notably, both of the above-mentioned assays require the use of protein-specific antibodies, which are not always available.

To overcome these technical limitations, researchers have developed several approaches that do not require cell extraction and direct protein handling. One approach is based on the establishment of auxotrophic yeast strains, obtained by the deletion of genes encoding essential metabolic enzymes. Such genes include HIS3, LEU2, LYS2 and TRP1, encoding for enzymes required for amino acid biosynthesis, as well as URA3 that encodes OMP decarboxylase (Ura3), an essential enzyme of pyrimidine ribonucleotide biosynthesis. Ura3 has been widely used in protein degradation studies. In these assays, constitutive expression of Ura3 rescues growth of ura3 cells in uracil-deficient medium¹³. Consequently, destabilizing Ura3 through the fusion of a degron can diminish cell growth on minimal medium lacking uracil. This method has been used in various protein degradation studies, including the identification of degradation determinants⁵, E3 ligases¹⁴ and auxiliary ubiquitylation factors¹⁵ and the discovery of novel UPS degrons¹⁶. All of these methods employed cell growth on agar plates as assay readout. However, the growth criterion (positive/negative growth), while robust and efficient, is mostly qualitative and does not provide quantitative information that is important for evaluating a degron's potency or the relative contribution of various auxiliary degradation factors.

We have therefore developed and utilized yeast vectors and screening methods enabling systematic and quantitative analysis of protein degradation by the URA3-degron fusion system. The protocol is based on an easy-to-handle assay that measures Growth kinetics in Liquid culture under Selective conditions (GiLS) and on the generation of standard growth curves. Yeast growth kinetics are characterized by three main phases — the lag, the exponential (log) and the stationary phase. Calculation of yeast replication kinetics during the log phase under selective conditions, which is determined by the levels of expression of the Ura3-degron, provides an unbiased quantitative measurement of protein degradation. This method can be applied to measuring and comparing degradation rates of multiple UPS substrates simultaneously in multiple strains and under various conditions.

Protocol

1. Cell Culture Transform the appropriate auxotrophic yeast cells, such as Try467 (Table 2), with a plasmid containing (a) a URA3-degron fusion and (b) an additional metabolic marker for plasmid selection and maintenance. NOTE: An example of a suitable plasmid is YDpK-MET25p-Deg1-FLAG-Vma12-Ura3 (LYS2) (Deg1-UV). This is a yeast integrative plasmid containing a fusion protein comprising of Deg1 — a degron derived from the yeast transcr…

Representative Results

Investigating the role of enzymes of the Doa10 pathway in the degradation of a reporter substrate To test the validity of the GiLS method it was compared to a traditional degradation assay. This experiment assesses the relative contribution of components of the ER-membrane localized Doa10 E3-ligase complex20,21 to the degradation of the protein quality control reporter substrate Deg1-VU (Figure 3A). The Deg1-VU plasmid was integra…

Discussion

Here we describe an assay based on cell growth for determining relative protein degradation rates, termed 'Growth kinetics in Liquid culture under Selective conditions' (GiLS). The GiLS assay has several advantages: It is simple to set up, data acquisition and analysis is straightforward and it is extremely modular. Consequently, GiLS can be applied simultaneously to multiple samples in a user friendly multi-well plate format that can be adapted to automation for high throughput applications. Most importantly, Gi…

Materials

Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Difco yeast nitrogen base w/o amino acids and ammonium sulfate	BD Biosciences	233520	For yeast growth on SD minimal media.  Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan
Ammonium sulfate	Sigma – Aldrich	A4418
Glucose	Sigma – Aldrich	16325
Adenine	Sigma – Aldrich	A8626
Uracil	Sigma – Aldrich	U0750
Amino acids	Highest purity available
96 well plates	Nunc	167008	Any other compatible brand can be used
Cyclohexamide	Sigma – Aldrich	C7698	Working conc. 0.5 mg/ml
Infinite 200 PRO series	Tecan		For yeast incubation and OD 600 measurements. Any other compatible temp-controled reader can be used
MDTcalc			Experimental software