Screening for interactions with predicted soluble domains of the yeast lactate ...

• Author(s): Pereira, Filipa ; Ribeiro, Gabriela ; Casal, Margarida ; Johansson, Björn ; Paiva, Sandra
• Date: 2011
• Persistent ID: http://hdl.handle.net/1822/15986
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Transporter; Protein interactions
• Document Type: Conference Object
• Language: English

Description

The S. cerevisiae membrane protein Jen1 is a monocarboxylate-proton symporter which after the addition of glucose to lactic acid-grown cells triggers loss of Jen1p activity and repression of JEN1 gene expression (Paiva et al., 2002; Andrade et al., 2001). This downregulation of the permease is dependent on phosphorylation and ubiquitylation mechanisms, both of which are dependent on proteins interacting with the Jen1p. To identify the proteins involved in this process we developed a system combined structure prediction and the yeast two-hybrid system to overcome the difficulties associated to measuring protein interactions with membrane proteins. Measuring protein interactions between membrane proteins or between membrane proteins and other proteins has proved to be very difficult. The commonly used yeast two-hybrid systems are inefficient for membrane proteins since the interaction must take place in the cell nucleus between soluble proteins. An advantage of the strategy used in our approach is that the screen is unbiased, i.e. contains all proteins in the yeast proteome. The structure of Jen1p predicts that it contains three cytosolic domains that would be expected participate in physiological protein interactions, important for regulation. We have cloned the three predicted cytosolic domains (1, 2 and 3) as bait fusions in the and screened a S. cerevisiae genomic library (James et al. 1996) after that we identified tree genes CIN5, PYC2 e SRO7. Based on this new approach, we hope to establish a reference library in yeast that may be used for many different purposes.