Fluorescent Proteins as Proteomic Probes

Cristea, Ileana M.; Williams, Rosemary; Chait, Brian T.; Rout, Michael P.

doi:10.1074/mcp.m500227-mcp200

Cited by 235 publications

(311 citation statements)

References 48 publications

Supporting

Mentioning

310

Contrasting

Order By: Relevance

“…In this strain (48), the chromosomal coding sequence for MukB has been replaced by GFP-tagged MukB, resulting in the expression of MukB-GFP under the control of the endogenous promoter. MukB-GFP-containing complexes were immunoprecipitated from cell lysates with a specific anti-GFP antibody (36). The isolated protein complexes for KAT1 and the wild-type W3110 control were subjected to SDS-PAGE and mass spectrometry (MS) analysis ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction

Stewart

Berger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

139

View full text Add to dashboard Cite

In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli , the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin. We demonstrate here a direct physical interaction between the dimerization domain of MukB and the C-terminal domain of the ParC subunit of Topo IV. In addition, we find that MukB alters the activity of Topo IV in vitro. Finally, we isolate a MukB mutant, D692A, that is deficient in its interaction with ParC and show that this mutant fails to rescue the temperature-sensitive growth phenotype of a mukB - strain. These results show that MukB and Topo IV are linked physically and functionally and indicate that the activities of these proteins are not limited to chromosome segregation but likely also play a key role in the control of higher-order bacterial chromosome structure.

show abstract

Section: Resultsmentioning

confidence: 99%

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction

Stewart

Berger

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

139

View full text Add to dashboard Cite

show abstract

“…One of the great advantages of GFP is its versatility as both a fluorescent marker and a very effective handle for the immunopurification of native complexes (16). The use of anti-GFP pull-downs for the high-resolution mapping of protein interactions by mass spectrometry is illustrated by recent studies using human lines containing GFP-tagged genes expressed on bacterial artificial chromosomes (17,18).…”

Section: A Scalable Strategy For the Knockin Of Gfp11 Repeats Enablesmentioning

confidence: 99%

“…Electroporation of RNP and ssDNA donor into cells results in very high (>30%) knockin efficiencies, whereas the limited RNP half-life in vivo minimizes off-target integration (14). We reasoned that this strategy would be well suited for large-scale knockin efforts in human cells and envisioned that GFP would be a functional tag of choice: on top of being a fluorescent marker, GFP is also a highly efficient purification handle for protein capture and subsequent proteomic analysis (16)(17)(18). GFP-tagged cells are also readily selectable by flow cytometry.…”

mentioning

confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

229

201

View full text Add to dashboard Cite

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...

show abstract

“…In the first method, an MTDH-eGFP fusion protein was created and stably overexpressed in LM2 cells. Whole cell lysates of LM2-MTDH-eGFP were subjected to immunoprecipitation with a highly specific anti-eGFP antibody (30) and separated by SDS-PAGE. Coomassie-stained bands (Fig.…”

Section: Identification Of Snd1 As An Mtdh-interacting Protein-tomentioning

confidence: 99%

“…Immunoprecipitation, Western Blot, and Immunofluorescence Analyses-For immunoprecipitation (IP) experiments, 80% confluent cells from 15-cm dishes were washed in cold PBS and lysed in 800 l of lysis buffer (same as used for mass spectrometry protein preparation) (30) with an EDTA-free protease inhibitor mixture (Roche Applied Science) and PMSF. Cell lysates were then sonicated and centrifuged.…”

mentioning

confidence: 99%

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions

Blanco

Alečković

Hua

et al. 2011

Journal of Biological Chemistry

Self Cite

100

114

View full text Add to dashboard Cite

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis.Metastasis is responsible for the majority of cancer-related deaths (1). An increasing number of genes have been implicated in mediating different steps of metastasis, but relatively few are mechanistically well characterized (2). One recently verified mediator of distant metastasis is Metadherin (MTDH 3 ; also called Lyric and AEG1 (3-5)). MTDH has been shown to be a key functional target of the 8q22 genomic gain that is frequently observed in poor prognosis breast cancer patients (6). Beyond promoting experimental lung metastasis (3, 6), multiple studies have implicated MTDH as a mediator of several cancer-related processes, such as oncogenesis and angiogenesis (7,8), invasion (9), chemoresistance (6, 10 -12), apoptosis resistance (13), and autophagy (14). Despite the abundance of MTDH phenotypes, a consensus understanding of its underlying molecular mechanisms has not yet been reached. MTDH has been shown to influence several oncogenic signaling pathways and transcription factors, such as Ha-Ras (15), PI3K/AKT (13), ERK, Wnt/␤-catenin (8), NF-B (16), c-Myc (15), and FOXO1/FOXO3a (17, 18). However, MTDH regulation of signaling pathways appears to be context-dependent with affected pathways varying by tumor type and cell line (19,20). Furthermore, prior work on MTDH molecular mechanisms has largely focused on hypothesis-driven investigations into the ability of MTDH to influence classical oncogenic pathways with little exploration to date on protein-level interactions (16,21).In this study, we aimed to expand the knowledge of MTDH molecular functionality and also uncover novel genes involved in promoting distant metastasis. Our approach utilized an unbiased, mass spectrometry-based screen for MTDH-interacting partners. We identified and characterized the interaction between MTDH and one such protein, SND1. SND1 is a multifunctional protein with reported roles in transcriptional activation (22), RNA editing (23, 24), formation of the RNAinduced ...

show abstract

Fluorescent Proteins as Proteomic Probes

Cited by 235 publications

References 48 publications

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions

Contact Info

Product

Resources

About