Proximity Labeling and Proteomics: Get to Know Neighbors.

Protein-protein interactions are essential in biological reactions and fundamental to cell-cell communication (e.g., the binding of secreted proteins, such as hormones, to cell membrane receptors) and the subsequent intracellular signal transduction cascade. Several studies have been extensively carried out on protein-protein interactions because they have the potential to resolve various problems in molecular biology. Biochemical methods, such as chemical cross-linking and immunoprecipitation, have long been used to analyze which proteins interact with each other. However, there are some problems, such as unphysiological states and non-specific binding, that require the development of more useful experimental methods. This chapter discusses the "proximity labeling (Proteomics)" analysis technique, which has been attracting attention in protein-protein interaction analysis in recent years and is used in many biological studies. "Membrane proximity labeling (proteomics)," which analyzes the interaction of cell membrane proteins, and "intracellular proximity labeling (proteomics)" will be explained in-depth.

The EMARS Reaction for Proximity Labeling.

To understand cellular processes at molecular levels, elucidation of protein-protein interactions occurring at a specific location in living cells is required. We have developed a proximity labeling method mediated by the enzyme-mediated activation of radical source (EMARS) reaction, which features a radical formation from labeling reagents by horseradish peroxidase (HRP) set on a molecule of interest (probed molecule). Proximal molecules are covalently labeled with a tag conjugated with the labeling reagent. Here we describe protocols for preparation of a labeling reagent, labeling of neighboring proteins of the probed molecule in living cells, and identification of the labeled proteins.

Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment.

We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000 bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a "gene cassette"). Then the various lengths of linear vectors (+0-+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500 bp at both ends of the gene cassette (+0-+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧50 bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5-300 fmol/shot), we observed that transformation efficiencies using 37.5 fmol (52.2 ng)/shot of the linear vector were not significantly different from those obtained using 300 fmol of the linear vector. The 300 fmol quantity was set considering the quantity of the circular plasmid (1 µg=approx. 300 fmol) and the 37.5 fmol quantity was set for quick and easy preparation of approximately 500 ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50 µl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications.

Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum.

Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

Each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal.

We previously reported a method, termed enzyme-mediated activation of radical sources (EMARS) for analysis of co-clustered molecules with horseradish peroxidase (HRP) fusion proteins expressed in living cells. This method is featured by radical formation of labeling reagents by HRP. In the current study, we have employed another labeling reagent, fluorescein-conjugated tyramide (FT) instead of the original arylazide compounds. Although hydrogen peroxide is required for the activation of FT, the labeling efficiency by HRP and the nonspecific reactions by endogenous enzyme(s) have been dramatically improved compared with the original fluorescein arylazide. This revised EMARS method has enabled visualization of co-clustered molecules in the endoplasmic reticulum and Golgi membranes with confocal microscopy. By using this method, we have found that GPI-anchored proteins, decay accelerating factor (DAF) and Thy-1 are exclusively co-clustered with HRP-DAFGPI and HRP-Thy1GPI, in which GPI attachment signals of DAF and Thy-1 have been connected to HRP, respectively. Furthermore, the N-glycosylation types of DAF and Thy-1 have been found to correspond to those of HRP-DAFGPI and HRP-Thy1GPI, respectively. These results indicate that each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal, and that the EMARS method can segregate individual lipid rafts.

Expressed glycosylphosphatidylinositol-anchored horseradish peroxidase identifies co-clustering molecules in individual lipid raft domains.

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.