Association of yeast SAP1, a novel member of the ‘AAA’ ATPase family of proteins, with the chromatin protein SIN1

Abstract

DNA in vitro [8]. Interestingly, sinl mutants are also defective Abstract The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional in proper segregation of chromosome III but not chromosome repressor of a family of genes. In addition, sinl mutants are V during mitosis [2]. We have shown in parallel work to that defective in proper mitotic chromosome segregation. In an effort described in this paper, that the N-terminal of SIN1 is capable to understand the basis for these phenotypes, we employed the of interaction with CDC23 [9], a protein known to control yeast two-hybrid system to identify proteins that interact with chromosome segregation during mitosis [10][11][12]. SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein Here we extend our previous studies to help explain the belonging to the 'AAA' family of ATPases, is able to directly biochemical basis for the disparate phenotypes of SIN1. We interact with SIN1. Furthermore, we show, using recombinant used the yeast two-hybrid system [13][14][15] as a method to molecules in vitro, that a short 27 amino acid sequence near the identify proteins that directly associate with SIN1. We demon-N-terminal of SIN1 is sufficient to bind SAP1. Previous strate that a short 27 amino acid sequence near the N-termiexperiments defined different domains of SIN that interact with other proteins and with DNA. The C-terminal domain of SIN1 nus of SIN1 is able to directly interact with a novel protein we was shown to be responsible for interaction with a protein that have termed SAP1 (SIN1 _associating protein). The predicted binds the regulatory region of HO, a gene whose transcription is amino acid sequence of SAP1 indicates that it is a member of repressed by SIN1. The central 'HMGl-like region' of SIN1 the 'AAA' (ATPase _associated with diverse cellular _activities) binds DNA, while the N-terminal of SIN1 can bind CDC23, a family of proteins. protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 2. Materials and methods is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different Plasmids and yeast strains cellular functions.Plasmid pBTM 116/SIN1 was constructed by subcloning the EcoRI fragment from pGEX-3X/SIN1 [16] containing the SIN1 coding re-

Functional Domains of the Yeast Chromatin Protein Sin1p/Spt2p Can Bind Four-way Junction and Crossing DNA Structures

“…For this purpose, we used two GSTSin1p fusion proteins containing amino acids 1-96 and 100 -162, engineered as described previously (10,25,26), each with a GST tag at the N terminus. Recombinant peptides are shown in Fig.…”

Functional characterization of fidgetin, an AAA-family protein mutated in fidget mice

“…MSP1 was localized to the cytosolic face of outer mitochondrial membrane with a putative role in mitochondrial protein sorting [26]. Finally, a minor family member SAP1 was shown to interact with a transcription repressor SIN1 in yeast [27]. SAP1 was proposed to be in the nucleus based on the putative involvement in transcription repression; however, it was recently shown to have a uniform cytoplasmic staining in yeast [28].…”

The C-Terminal Domain of Sin1 Interacts with the SWI-SNF Complex in Yeast

“…A likely possibility is that Sin1 that is not complexed in chromatin is degraded. It has been reported that the Nt domain of Sin1 interacts with Cdc23 (37), a component of the APC ubiquitin ligase and a protein with homologies to the AAA family of proteasome components (19). It is possible that these factors affect the stability of Sin1.…”