Extraction of cell surface-associated proteins from living yeast cells.

To extract cell surface-associated proteins from living fungal cells, reducing agents such as beta-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especially at high extraction temperatures. To avoid artifacts, we recommend using (a) a low concentration of the reducing agent for only a short period of time, and (b) an extraction temperature of 4 degrees C to protect the integrity of the plasma membrane.

Dissection of the promoter of the HAP4 gene in S. cerevisiae unveils a complex regulatory framework of transcriptional regulation.

In S. cerevisiae, the heteromeric Hap2/3/4/5 complex is necessary for induced transcription of a large number of genes involved in oxidative metabolism on non-fermentable carbon sources. The Hap4p subunit is the activator subunit and at the same time also the regulatory part of the complex, since it is the only one whose level is regulated by carbon source itself.