Uncoupling the Functions of CALM in VAMP Sorting and Clathrin-Coated Pit Formation

Daniela A Sahlender,Patrycja Kozik,Sharon E Miller,Andrew A Peden,Margaret S Robinson,Julie G Donaldson

doi:10.1371/journal.pone.0064514

Daniela A Sahlender, Patrycja Kozik + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0064514

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Journal: PloS one	Publication Date: May 31, 2013
Citations: 21	License type: CC BY 4.0

Affiliation: University of Cambridge

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.

Highlights

Proteins belonging to the CALM/AP180 family are found in most eukaryotes, and are major components of the coats on endocytic clathrin-coated vesicles (CCVs)
When the cells are analysed by flow cytometry, the green-to-red ratio gives a measure of endocytosis efficiency. b, Western blot of homogenates of control and siRNA-treated cells, probed for CALM, HA-tagged VAMP2 (V2-HA), and syntaxin 4 (Stx4)
Endocytosis Efficiency Assay To monitor R-SNARE sorting in different cell lines under different conditions, we needed an assay that would not be affected by variations in expression level

Summary

Introduction

Proteins belonging to the CALM/AP180 family are found in most eukaryotes, and are major components of the coats on endocytic clathrin-coated vesicles (CCVs). Knocking out the two redundant family members in Saccharomyces cerevisiae, Yap1801 and Yap1802, profoundly affects the internalization of the R-SNARE Snc, without affecting the internalization of other CCV cargo proteins [3]; and knocking down CALM in human embryonic kidney cells causes transiently transfected VAMP2, another R-SNARE, to accumulate on the plasma membrane [4]. Together, these studies suggested that CALM might be an adaptor for certain types of RSNAREs, even though for many years no physical interactions were reported

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