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Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Borner GH, Hein MY, Hirst J, Edgar JR, Mann M, Robinson MS - Mol. Biol. Cell (2014)

Bottom Line: Functionally associated groups of proteins are revealed through cluster analysis.Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes.In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany borner@biochem.mpg.de.

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Application of fractionation profiling to S2 cells reveals the composition of Drosophila CCVs. (A) Drosophila S2 lysates were subfractionated as in Figure 1A. Gel electrophoresis (Coomassie stain) reveals that all subfractions have different compositions. The probable clathrin heavy chain band is indicated (arrow). (B) Western blotting of fractions shown in A confirms that CHC and the associated adaptor AP-1 have similar profiles, which are distinct from the nonclathrin adaptor AP-3. (C) Proteomic analysis of S2 fractions from two independent profiling experiments identified 1799 proteins with complete profiles. PCA of the profiles reveals clustering of subunits of known protein complexes, including AP-1, AP-2, AP-3, the anaphase-promoting complex (APC), the T-complex (CCT), clathrin heavy and light chains (Cla), the Exocyst (Exo), the mitochondrial ribosome (mRP), the proteasome core (PS20) and regulatory particle (PS19), signalosome (Sig), and the V-ATPase (vATP0, integral membrane subcomplex; vATP1, peripheral subcomplex). Of importance, proteins whose mammalian homologues are known CCV proteins cluster in the vicinity of clathrin (marked in red, CCV). Fractionation profiling successfully reveals the composition of the Drosophila CCV proteome. (x-, y-axes = first and second principal components; cumulative R2 = 0.917). To illustrate that even a single fractionation profiling experiment is sufficient to produce meaningful clustering, this plot shows PCA of the first triplet only (i.e., three data points). Joined PCA of both triplets (all six data points) results in a very similar plot. (D) Four candidate Drosophila CCV coat components predicted by fractionation profiling were C-terminally tagged and transiently expressed in S2 cells. Proteins were visualized by immunofluorescence microscopy. All four proteins colocalize with established markers of clathrin-coated vesicles, confirming their association with CCVs in S2 cells. LqfR, SCYL2, and SMAP2 colocalize tightly with AP-1, a marker of TGN/endosomal CCVs. SES1/2 localizes to plasma membrane clathrin-coated pits. Because clathrin light chain (CLC) marks both endocytic and intracellular clathrin-coated structure, it shows only partial colocalization with SES1/2. Some areas of colocalization are indicated (white arrowheads). Scale bar, 10 μm.
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Figure 3: Application of fractionation profiling to S2 cells reveals the composition of Drosophila CCVs. (A) Drosophila S2 lysates were subfractionated as in Figure 1A. Gel electrophoresis (Coomassie stain) reveals that all subfractions have different compositions. The probable clathrin heavy chain band is indicated (arrow). (B) Western blotting of fractions shown in A confirms that CHC and the associated adaptor AP-1 have similar profiles, which are distinct from the nonclathrin adaptor AP-3. (C) Proteomic analysis of S2 fractions from two independent profiling experiments identified 1799 proteins with complete profiles. PCA of the profiles reveals clustering of subunits of known protein complexes, including AP-1, AP-2, AP-3, the anaphase-promoting complex (APC), the T-complex (CCT), clathrin heavy and light chains (Cla), the Exocyst (Exo), the mitochondrial ribosome (mRP), the proteasome core (PS20) and regulatory particle (PS19), signalosome (Sig), and the V-ATPase (vATP0, integral membrane subcomplex; vATP1, peripheral subcomplex). Of importance, proteins whose mammalian homologues are known CCV proteins cluster in the vicinity of clathrin (marked in red, CCV). Fractionation profiling successfully reveals the composition of the Drosophila CCV proteome. (x-, y-axes = first and second principal components; cumulative R2 = 0.917). To illustrate that even a single fractionation profiling experiment is sufficient to produce meaningful clustering, this plot shows PCA of the first triplet only (i.e., three data points). Joined PCA of both triplets (all six data points) results in a very similar plot. (D) Four candidate Drosophila CCV coat components predicted by fractionation profiling were C-terminally tagged and transiently expressed in S2 cells. Proteins were visualized by immunofluorescence microscopy. All four proteins colocalize with established markers of clathrin-coated vesicles, confirming their association with CCVs in S2 cells. LqfR, SCYL2, and SMAP2 colocalize tightly with AP-1, a marker of TGN/endosomal CCVs. SES1/2 localizes to plasma membrane clathrin-coated pits. Because clathrin light chain (CLC) marks both endocytic and intracellular clathrin-coated structure, it shows only partial colocalization with SES1/2. Some areas of colocalization are indicated (white arrowheads). Scale bar, 10 μm.

Mentions: Although Drosophila is a widely used model organism, the composition of its CCVs remains poorly characterized. To test whether fractionation profiling is transferable to other cell systems, we chose to investigate Drosophila S2 cells. We applied fractionation profiling without further optimization (Figure 3A). Clathrin and associated proteins had profiles similar to those in HeLa cells (Figure 3B). For proteomic analysis, we prepared a reference and three subfractions from SILAC-labeled S2 cells, repeated the preparation with reversed labeling, and analyzed the six sample pairs by mass spectrometry. In total, we identified >3000 proteins, of which 1799 were quantified across all six samples. PCA shows that subunits of known protein complexes form clusters, as expected (Figure 3C). Known fly CCV proteins, including clathrin, AP-1, and AP-2, formed a distinct cluster in the periphery of the plot. We then constructed a Predictor for the S2 profiling data (Supplemental Table S3). A search against Drosophila clathrin heavy chain revealed a list of 29 candidate CCV proteins predicted with the highest level of confidence (Table 1). Remarkably, the human homologues of 27 of these are known CCV proteins; most of these proteins have not been characterized in Drosophila. An extended search with lower stringency revealed up to 50 candidate CCV proteins (Supplemental Table S4). To validate some of our predictions, we tagged four candidate coat proteins (Figure 3D). All four showed extensive colocalization with established markers of CCVs. In sum, fractionation profiling was successfully implemented to characterize CCVs from S2 cells. Given the evolutionary distance between humans and Drosophila, it is highly likely that the approach will be applicable to a wide variety of cell types.


Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Borner GH, Hein MY, Hirst J, Edgar JR, Mann M, Robinson MS - Mol. Biol. Cell (2014)

Application of fractionation profiling to S2 cells reveals the composition of Drosophila CCVs. (A) Drosophila S2 lysates were subfractionated as in Figure 1A. Gel electrophoresis (Coomassie stain) reveals that all subfractions have different compositions. The probable clathrin heavy chain band is indicated (arrow). (B) Western blotting of fractions shown in A confirms that CHC and the associated adaptor AP-1 have similar profiles, which are distinct from the nonclathrin adaptor AP-3. (C) Proteomic analysis of S2 fractions from two independent profiling experiments identified 1799 proteins with complete profiles. PCA of the profiles reveals clustering of subunits of known protein complexes, including AP-1, AP-2, AP-3, the anaphase-promoting complex (APC), the T-complex (CCT), clathrin heavy and light chains (Cla), the Exocyst (Exo), the mitochondrial ribosome (mRP), the proteasome core (PS20) and regulatory particle (PS19), signalosome (Sig), and the V-ATPase (vATP0, integral membrane subcomplex; vATP1, peripheral subcomplex). Of importance, proteins whose mammalian homologues are known CCV proteins cluster in the vicinity of clathrin (marked in red, CCV). Fractionation profiling successfully reveals the composition of the Drosophila CCV proteome. (x-, y-axes = first and second principal components; cumulative R2 = 0.917). To illustrate that even a single fractionation profiling experiment is sufficient to produce meaningful clustering, this plot shows PCA of the first triplet only (i.e., three data points). Joined PCA of both triplets (all six data points) results in a very similar plot. (D) Four candidate Drosophila CCV coat components predicted by fractionation profiling were C-terminally tagged and transiently expressed in S2 cells. Proteins were visualized by immunofluorescence microscopy. All four proteins colocalize with established markers of clathrin-coated vesicles, confirming their association with CCVs in S2 cells. LqfR, SCYL2, and SMAP2 colocalize tightly with AP-1, a marker of TGN/endosomal CCVs. SES1/2 localizes to plasma membrane clathrin-coated pits. Because clathrin light chain (CLC) marks both endocytic and intracellular clathrin-coated structure, it shows only partial colocalization with SES1/2. Some areas of colocalization are indicated (white arrowheads). Scale bar, 10 μm.
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Figure 3: Application of fractionation profiling to S2 cells reveals the composition of Drosophila CCVs. (A) Drosophila S2 lysates were subfractionated as in Figure 1A. Gel electrophoresis (Coomassie stain) reveals that all subfractions have different compositions. The probable clathrin heavy chain band is indicated (arrow). (B) Western blotting of fractions shown in A confirms that CHC and the associated adaptor AP-1 have similar profiles, which are distinct from the nonclathrin adaptor AP-3. (C) Proteomic analysis of S2 fractions from two independent profiling experiments identified 1799 proteins with complete profiles. PCA of the profiles reveals clustering of subunits of known protein complexes, including AP-1, AP-2, AP-3, the anaphase-promoting complex (APC), the T-complex (CCT), clathrin heavy and light chains (Cla), the Exocyst (Exo), the mitochondrial ribosome (mRP), the proteasome core (PS20) and regulatory particle (PS19), signalosome (Sig), and the V-ATPase (vATP0, integral membrane subcomplex; vATP1, peripheral subcomplex). Of importance, proteins whose mammalian homologues are known CCV proteins cluster in the vicinity of clathrin (marked in red, CCV). Fractionation profiling successfully reveals the composition of the Drosophila CCV proteome. (x-, y-axes = first and second principal components; cumulative R2 = 0.917). To illustrate that even a single fractionation profiling experiment is sufficient to produce meaningful clustering, this plot shows PCA of the first triplet only (i.e., three data points). Joined PCA of both triplets (all six data points) results in a very similar plot. (D) Four candidate Drosophila CCV coat components predicted by fractionation profiling were C-terminally tagged and transiently expressed in S2 cells. Proteins were visualized by immunofluorescence microscopy. All four proteins colocalize with established markers of clathrin-coated vesicles, confirming their association with CCVs in S2 cells. LqfR, SCYL2, and SMAP2 colocalize tightly with AP-1, a marker of TGN/endosomal CCVs. SES1/2 localizes to plasma membrane clathrin-coated pits. Because clathrin light chain (CLC) marks both endocytic and intracellular clathrin-coated structure, it shows only partial colocalization with SES1/2. Some areas of colocalization are indicated (white arrowheads). Scale bar, 10 μm.
Mentions: Although Drosophila is a widely used model organism, the composition of its CCVs remains poorly characterized. To test whether fractionation profiling is transferable to other cell systems, we chose to investigate Drosophila S2 cells. We applied fractionation profiling without further optimization (Figure 3A). Clathrin and associated proteins had profiles similar to those in HeLa cells (Figure 3B). For proteomic analysis, we prepared a reference and three subfractions from SILAC-labeled S2 cells, repeated the preparation with reversed labeling, and analyzed the six sample pairs by mass spectrometry. In total, we identified >3000 proteins, of which 1799 were quantified across all six samples. PCA shows that subunits of known protein complexes form clusters, as expected (Figure 3C). Known fly CCV proteins, including clathrin, AP-1, and AP-2, formed a distinct cluster in the periphery of the plot. We then constructed a Predictor for the S2 profiling data (Supplemental Table S3). A search against Drosophila clathrin heavy chain revealed a list of 29 candidate CCV proteins predicted with the highest level of confidence (Table 1). Remarkably, the human homologues of 27 of these are known CCV proteins; most of these proteins have not been characterized in Drosophila. An extended search with lower stringency revealed up to 50 candidate CCV proteins (Supplemental Table S4). To validate some of our predictions, we tagged four candidate coat proteins (Figure 3D). All four showed extensive colocalization with established markers of CCVs. In sum, fractionation profiling was successfully implemented to characterize CCVs from S2 cells. Given the evolutionary distance between humans and Drosophila, it is highly likely that the approach will be applicable to a wide variety of cell types.

Bottom Line: Functionally associated groups of proteins are revealed through cluster analysis.Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes.In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany borner@biochem.mpg.de.

Show MeSH
Related in: MedlinePlus