Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Summary

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Abstract

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Protocol

A. Cell Culture and Protein Induction

This section describes cell culture conditions, cell-cycle synchronization with Nocodazole, followed by induction of protein expression from a regulatable promoter in yeast cells.

1. The wild type yeast strain W303 (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) is transformed with high copy (2μ) plasmid DNA that contains our gene of interest (ENTH2) under the control of a methionine-repressible promoter (MET25). 2mM methionine is sufficient to suppress promoter activity, while media lacking methionine allows maximal expression. Hence, a range of methionine concen....

Discussion

This protocol describes how to monitor the development of a morphological phenotype (yeast cell unable to undergo proper cell division) upon protein overexpression. When doing this procedure it's important to remember to harvest yeast cells by pelleting at the recommended centrifugation speed as faster speeds may damage cells and obscure results. Methylene blue and Calcofluor white should be added to live cells just prior to imaging as they are toxic. This procedure can also be easily adapted for phenotypes observed unde.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nocodazole	Reagent	Sigma-Aldrich	M1404
Methylene Blue 1% (w/v)	Reagent	VWR international	VW6276-0
Calcofluor White	Reagent	Sigma-Aldrich	F3543
Petroleum Jelly	Reagent	VWR international	VW3339-2