Kernimport des TEAD-Transkriptionsfaktors Tec1 aus Saccharomyces cerevisiae

Nuclear transport of the TEAD-familiy transcription factors has not been studied so far. In this work, nuclear import of Tec1, the only TEAD-protein from Saccharomyces cerevisiae, has been investigated in detail. In S. cerevisiae, Tec1 fulfills versatile functions by controlling cell division and cell differentiation. The study revealed that two regions of the protein, NLS1 and NLS2 (NLS for nuclear localization signal), are necessary and sufficient for nuclear localization of Tec1. NLS1 lies in the N-terminal half of the protein and overlaps completely with the TEA-domain which confers DNA-binding. Several amino acids were identified to be critical for the function of NLS1 and that most of these residues are also important for binding to TCS-elements (TCS for TEA consensus sequence) in the DNA. In a structural model, these residues are arranged in the same plane, indicating that this area serves as the contact site for DNA-binding as well as probably for importins. Three amino acids have been identified, whose side chains are not crucial for DNA-binding, but mutation of these residues leads to a decreased efficiency of nuclear import. Therefore, the functions DNA-binding and nuclear localization can be separated at least partially. NLS2 is in the C-terminal half of Tec1 and corresponds to a region which is necessary for interactions with other proteins, for example the transcription factor Ste12. It could be shown that nuclear import of Tec1 is not affected in the absence of Ste12. In NLS2 two adjacent residues, lysine 318 and arginine 319, have been identified to be crucial for its function. Further genetic and cell biological analyses showed that both NLS-regions are required for optimal nuclear import. Biochemical binding assays revealed that both NLSs can interact with several importins. Three of them can recognize both localization signals, whereas four importins are specific for NLS1 or NLS2. According to the results of this work, there are numerous ways for Tec1 nuclear import, because it contains two NLSs which interact with several importins each. This leads to the conclusion that nuclear import of Tec1 is ensured under different conditions so that this TEAD-transcription factor can fulfill its versatile functions.