CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature

Summary

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

Graphical Abstract

Highlights

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  CALM loss increases size and frequency of early endocytic clathrin-coated structures

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  Depletion of CALM slows endocytic clathrin-coated pit maturation and endocytic rate

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  CALM possesses an N-terminal, membrane-curvature-sensing/driving amphipathic helix

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  Clathrin-coated pit maturation is regulated by CALM’s N-terminal amphipathic helix

Abstract

Miller et al. demonstrate that depletion of the abundant endocytic protein CALM increases the diameter and percentage of early endocytic clathrin-coated structures (CCSs) while delaying CCS maturation and reducing endocytic rates. CALM’s ability to influence these CCS properties depends on a membrane-inserting amphipathic helix, which senses and promotes membrane curvature.