Sahlender, D.A., Kozik, P., Miller, S.E. et al. (2 more authors) (2013) Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation. PLoS One, 8 (5). e64514. ISSN 1932-6203
Abstract
CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2013 Sahlender et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
Keywords: | Amino Acid Sequence; Clathrin; Coated Pits, Cell-Membrane; Endocytosis; Gene Expression; Golgi Apparatus; HeLa Cells; Humans; Microscopy, Fluorescence; Monomeric Clathrin Assembly Proteins; Mutation; Protein Sorting Signals; Protein Transport; R-SNARE Proteins; RNA, Small Interfering; Sequence Alignment; Signal Transduction; Transport Vesicles |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Biomedical Science (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 20 Dec 2016 13:53 |
Last Modified: | 20 Dec 2016 13:53 |
Published Version: | http://dx.doi.org/10.1371/journal.pone.0064514 |
Status: | Published |
Publisher: | Public Library of Science |
Refereed: | Yes |
Identification Number: | 10.1371/journal.pone.0064514 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:108289 |