title: An in vivo photo-cross-linking approach reveals a homodimerization domain of Aha1 in S. cerevisiae.
abstract: Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo.
authors.affiliation: Fraunhofer IGB, Stuttgart, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Berg
authors.affiliation: IBVT, University of Stuttgart, Stuttgart, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Michalowski
authors.affiliation: IGVP, University of Stuttgart, Stuttgart, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Palzer
authors.affiliation: Fraunhofer IGB, Stuttgart, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Rupp
authors.affiliation: Fraunhofer IGB, Stuttgart, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Sohn
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000528:radiation effects*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: C475092:AHA1 protein, S cerevisiae; D001386:Azides; D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C027162:4-azidophenylalanine; D010649:Phenylalanine; D018833:Chaperonins