Min-Kyung Sung

Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae.

The bimolecular fluorescence complementation (BiFC) assay has been widely accepted for studying in vivo detection of protein–protein interactions in several organisms. To facilitate the application of the BiFC assay to yeast research, we have created a series of plasmids that allow single‐step, PCR‐based C‐ or N‐terminal tagging of yeast proteins with yellow fluorescent protein fragments for BiFC assay. By examination of several interacting proteins (Sis1–Sis1, Net1–Sir2, Cet1–Cet1 and Pho2–Pho4), we demonstrate that the BiFC assay can be used to reliably analyse the occurrence and subcellular localization of protein–protein interactions in living yeast cells. The sequences for the described plasmids were submitted to the GenBank under Accession Nos: EF210802, pFA6a‐VN‐His3MX6; EF210803, pFA6a‐VC‐His3MX6; EF210804, pFA6a‐VN‐TRP1; EF210807, pFA6a‐VC‐TRP1; EF210808, pFA6a‐VN‐kanMX6; EF210809, pFA6a‐VC‐kanMX6; EF210810, pFA6a‐His3MX6‐PGAL1‐VN; EF210805, pFA6a‐His3MX6‐PGAL1‐VC; EF210806, pFA6a‐TRP1‐PGAL1‐VN; EF210811, pFA6a‐TRP1‐PGAL1‐VC; EF210812, pFA6a‐kanMX6‐PGAL1‐VN; EF210813, pFA6a‐kanMX6‐PGAL1‐VC; EF521883, pFA6a‐His3MX6‐PCET1‐VN; EF521884, pFA6a‐His3MX6‐PCET1‐VC; EF521885, pFA6a‐TRP1‐PCET1‐VN; EF521886, pFA6a‐TRP1‐PCET1‐VC; EF521887, pFA6a‐kanMX6‐PCET1‐VN; EF521888, pFA6a‐kanMX6‐PCET1‐VC. Copyright

Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast

The definition of protein-protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.

A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, one‐step PCR‐mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C‐ or N‐terminus. For many purposes, C‐terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C‐terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module‐based C‐terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C‐terminal epitope tagging of proteins in yeast cells that already carry MX6 module‐based gene deletion or C‐terminal epitope tag. Copyright

Nsi1 plays a significant role in the silencing of ribosomal DNA in Saccharomyces cerevisiae

In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100–200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay.

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.

Proteome-wide remodeling of protein location and function by stress

Significance Protein location and function are dependent on diverse cell states. We develop a conditional function predictor (CoFP) for proteome-wide prediction of condition-specific locations and functions of proteins. In addition to highly accurate retrieval of condition-dependent locations and functions in individual conditions, CoFP successfully discovers dynamic function changes of yeast proteins, including Tsr1, Caf120, Dip5, Skg6, Lte1, and Nnf2, under DNA-damaging stresses. Beyond specific predictions, CoFP reveals a global landscape of changes in protein location and function, highlighting a surprising number of proteins that translocate from the mitochondria to the nucleus or from endoplasmic reticulum to Golgi apparatus under stress. CoFP has the potential to discover previously unidentified condition-specific locations and functions under diverse conditions of cellular growth. Protein location and function can change dynamically depending on many factors, including environmental stress, disease state, age, developmental stage, and cell type. Here, we describe an integrative computational framework, called the conditional function predictor (CoFP; http://nbm.ajou.ac.kr/cofp/), for predicting changes in subcellular location and function on a proteome-wide scale. The essence of the CoFP approach is to cross-reference general knowledge about a protein and its known network of physical interactions, which typically pool measurements from diverse environments, against gene expression profiles that have been measured under specific conditions of interest. Using CoFP, we predict condition-specific subcellular locations, biological processes, and molecular functions of the yeast proteome under 18 specified conditions. In addition to highly accurate retrieval of previously known gold standard protein locations and functions, CoFP predicts previously unidentified condition-dependent locations and functions for nearly all yeast proteins. Many of these predictions can be confirmed using high-resolution cellular imaging. We show that, under DNA-damaging conditions, Tsr1, Caf120, Dip5, Skg6, Lte1, and Nnf2 change subcellular location and RNA polymerase I subunit A43, Ino2, and Ids2 show changes in DNA binding. Beyond specific predictions, this work reveals a global landscape of changing protein location and function, highlighting a surprising number of proteins that translocate from the mitochondria to the nucleus or from endoplasmic reticulum to Golgi apparatus under stress.