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Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking.

Hirst J, Edgar JR, Borner GH, Li S, Sahlender DA, Antrobus R, Robinson MS - Mol. Biol. Cell (2015)

Bottom Line: The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown.Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins.Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Proteomics of CCVs from SILAC-labeled epsinR knocksideways cells. The fold change (control:knocksideways) was calculated for each protein, and proteins were ranked from the highest to lowest ratio. Proteins with ratios of ≥2.0 are shown in order of rank for the epsinR knocksideways. For ease of visualization, known CCV components are color coded.
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Figure 2: Proteomics of CCVs from SILAC-labeled epsinR knocksideways cells. The fold change (control:knocksideways) was calculated for each protein, and proteins were ranked from the highest to lowest ratio. Proteins with ratios of ≥2.0 are shown in order of rank for the epsinR knocksideways. For ease of visualization, known CCV components are color coded.

Mentions: To look for changes in the overall protein composition of CCVs in epsinR knocksideways cells, we carried out stable isotope labeling by amino acids in cell culture (SILAC)–based mass spectrometry on CCV fractions from control and rapamycin-treated cells after first depleting endogenous epsinR with siRNA. Figure 2 shows a list of proteins depleted twofold or more from the knocksideways CCVs, based on three independent experiments, with known CCV components indicated in color. We had anticipated that with the exception of epsinR itself and vti1b, most coat and cargo proteins would be relatively unaffected. Surprisingly, however, the epsinR knocksideways had a global effect on intracellular CCVs, similar to the effect of an AP-1 knocksideways. The top hits included not only epsinR, but also GGA2, AP-1, and clathrin, indicating that, like AP-1, epsinR normally acts as a linchpin for the formation of an entire population of CCVs. Moreover, although vti1b was depleted from the preparation, several cargo proteins that depend on AP-1 and/or GGAs (e.g., hydrolase receptors and carboxypeptidase D) were depleted even more strongly. It is not clear why the abundant intracellular clathrin-coated structures we observed by electron microscopy were not recovered in our CCV fraction, but the altered morphology is consistent with some sort of abnormality in their ability to dissociate from membranes.


Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking.

Hirst J, Edgar JR, Borner GH, Li S, Sahlender DA, Antrobus R, Robinson MS - Mol. Biol. Cell (2015)

Proteomics of CCVs from SILAC-labeled epsinR knocksideways cells. The fold change (control:knocksideways) was calculated for each protein, and proteins were ranked from the highest to lowest ratio. Proteins with ratios of ≥2.0 are shown in order of rank for the epsinR knocksideways. For ease of visualization, known CCV components are color coded.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551321&req=5

Figure 2: Proteomics of CCVs from SILAC-labeled epsinR knocksideways cells. The fold change (control:knocksideways) was calculated for each protein, and proteins were ranked from the highest to lowest ratio. Proteins with ratios of ≥2.0 are shown in order of rank for the epsinR knocksideways. For ease of visualization, known CCV components are color coded.
Mentions: To look for changes in the overall protein composition of CCVs in epsinR knocksideways cells, we carried out stable isotope labeling by amino acids in cell culture (SILAC)–based mass spectrometry on CCV fractions from control and rapamycin-treated cells after first depleting endogenous epsinR with siRNA. Figure 2 shows a list of proteins depleted twofold or more from the knocksideways CCVs, based on three independent experiments, with known CCV components indicated in color. We had anticipated that with the exception of epsinR itself and vti1b, most coat and cargo proteins would be relatively unaffected. Surprisingly, however, the epsinR knocksideways had a global effect on intracellular CCVs, similar to the effect of an AP-1 knocksideways. The top hits included not only epsinR, but also GGA2, AP-1, and clathrin, indicating that, like AP-1, epsinR normally acts as a linchpin for the formation of an entire population of CCVs. Moreover, although vti1b was depleted from the preparation, several cargo proteins that depend on AP-1 and/or GGAs (e.g., hydrolase receptors and carboxypeptidase D) were depleted even more strongly. It is not clear why the abundant intracellular clathrin-coated structures we observed by electron microscopy were not recovered in our CCV fraction, but the altered morphology is consistent with some sort of abnormality in their ability to dissociate from membranes.

Bottom Line: The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown.Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins.Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.

No MeSH data available.


Related in: MedlinePlus