Membrane orientation of small multidrug resistance proteins

PhD ceremony: Ms. M.A. Kolbusz, 11.00 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Membrane orientation of small multidrug resistance proteins

Promotor(s): prof. D.J. Slotboom, prof. A.J.M. Driessen

Faculty: Mathematics and Natural Sciences

The thesis of Magdalena Anna Kolbusz focuses on membrane orientation of members of the Small Multidrug Resistance (SMR) protein family and its correlation with distribution of positively charged amino acid residues over the membrane. As described in the positive-inside rule the positively charged residues are located mostly in cytoplasmic loops and seem to bethe key orientation determinant. In the SMR family there are two types of proteins: singlesandpairs encoded by single and paired genes respectively. The EmrE protein, which belongs to the singles, inserts into the membrane in two opposite orientations (dual topology). To verify the orientation of other SMR proteins membrane orientation of 9 singles and 9 pairs was established. It has been shown that singlesacquire dual topology as EmrE. Analysis of distribution of positively charged amino acids showed that there is a good correlation between the orientation and the distribution of positively charged residues, but also exceptions violating the positive-inside rule have been found. Among pairs, proteins inserted into the membrane in fixed, opposite orientations in agreement with the distribution of positively charged amino acids. A detailed analysis ofthe role of charged residues on orientation showed that different residues influence orientation with different strength and probably there are other factors involved in orientation determination.

Analysis of 29 Escherichia colii strains revealed that in 10 strains a longer version of EmrE is present. The elongated protein contains 5 TMSs and acquires a single membrane orientation. Its function remains unknown and requires further research.