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Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

ABSTRACT: The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

SUBMITTER: Caceres PS

PROVIDER: S-EPMC6575629 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

Caceres Paulo S PS Gravotta Diego D Zager Patrick J PJ Dephoure Noah N Rodriguez-Boulan Enrique E

Proceedings of the National Academy of Sciences of the United States of America 20190529 24

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope label ...[more]

PMID: 31142645

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Project description:BackgroundThe notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.Methodology/principal findingsWe report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37 degrees C. If cells are first incubated with LDL at 4 degrees C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37 degrees C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway.Conclusions/significanceThese results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.

Dataset Information

Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

Publications

Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

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