Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Summary

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).

Abstract

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.

Introduction

Proteins perform crucial functions in virtually every cellular process. Many physiological processes require the presence of a specific protein (or proteins) for a defined period of time or under particular circumstances. Organisms therefore monitor and regulate protein abundance to meet cellular needs ¹. For example, cyclins (proteins that control cell division) are present at specific phases of the cell cycle, and the loss of regulated cyclin levels has been associated with malignant tumor formation ². In addition to regulating protein levels to meet cellular needs, cells employ degradative quality control mechanisms to eliminate misfolded, una....

Protocol

1. Growth and Harvest of Yeast Cells

1. If not analyzing degradation kinetics of an endogenous yeast protein, transform desired yeast strain(s) with a plasmid encoding the protein of interest. Reliable methods for yeast transformation have been previously described ³⁷.
2. Inoculate yeast in 5 ml of appropriate medium (e.g., selective synthetic defined (SD) medium for plasmid maintenance of transformed cells or non-selective yeast extract-peptone-dextrose (YPD) medium for non-transformed cells). Incubate overnight at 30 °C, rotating.
   NOTE: 30 °C is the optimal growth temperature for typical wild-type laboratory yeast....

Results

To illustrate cycloheximide chase methodology, the stability of Deg1-Sec62 (Figure 1), a model yeast endoplasmic reticulum (ER)-associated degradation (ERAD) substrate, was analyzed ^42-44. In ERAD, quality control ubiquitin ligase enzymes covalently attach chains of the small protein ubiquitin to aberrant proteins localized to the ER membrane. Such polyubiquitylated proteins are subsequently removed from the ER and degraded by the proteasome, a large, .......

Discussion

In this paper, a method for analyzing protein degradation kinetics is presented. This technique can be readily applied to a range of proteins degraded by a variety of mechanisms. It is important to note that cycloheximide chase experiments report on degradation kinetics of the steady state pool of a given protein. Other techniques may be used to analyze the degradation kinetics of specific populations of proteins. For example, the degradative fate of nascent polypeptides can be tracked by pulse chase analysis ⁵⁵

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Desired yeast strains, plasmids, standard medium and buffer components
Disposable borosilicate glass tubes	Fisher Scientific	14-961-32	Available from a variety of manufacturers
Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180)	Dot Scientific	51028068	Available from a variety of manufacturers
New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)	New Brunswick	M1053-0450	A tube roller is recommended to maintain overnight  starter cultures of yeast cells in suspension. Alternatively, if a tube roller is unavailable, a platform shaker in a temperature-controlled incubator may be used for overnight starter cultures. A platform shaker or tube roller may be used to maintain larger cultures in suspension.
New Brunswick TC-7 Roller Drum 120 V 50/60 H	New Brunswick	M1053-4004	For use with tube roller
SmartSpec Plus Spectrophotometer	Bio-Rad	170-2525	Available from a variety of manufacturers
Centrifuge 5430	Eppendorf	5427 000.216	Rotor that is sold with unit holds 1.5 and 2.0 ml microcentrifuge tubes. Rotor may be swapped for one that holds 15 and 50 ml conical tubes
Fixed-Angle Rotor F-35-6-30 with Lid and Adapters for Centrifuge Model 5430/R, 15/50 ml Conical Tubes, 6-Place	Eppendorf	F-35-6-30
15-ml screen printed screw cap tube 17 x 20 mm conical, polypropylene	Sarstedt	62.554.205	Available from a variety of manufacturers
1.5-ml flex-tube, PCR clean, natural microcentrifuge tubes	Eppendorf	22364120	Available from a variety of manufacturers
Analog Dri-Bath Heaters	Fisher Scientific	1172011AQ	It is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 °C water bath may be used for incubation of cells in the presence of cycloheximide. Boiling water bath with hot plate may altertnatively be used to denature proteins.
Heating block for 12 x 15 ml conical tubes	Fisher Scientific	11-473-70	For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.
Heating block for 20 x 1.5 ml conical tubes	Fisher Scientific	11-718-9Q	For use with Dri-Bath Heater to boil lysates for protein denaturation.
SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane			Will vary by lab and application
Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software)	Li-Cor	9140-01	Will vary by lab and application
EMD Millipore Immobilon PVDF Transfer Membranes	Fisher Scientific	IPFL00010	Will vary by lab and application
Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))	Life Technologies	459250	Will vary by lab and application
Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H + L))	Life Technologies	A-21065	Will vary by lab and application