A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1

Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors...

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Bibliographic Details
Published in:Biochemistry (Easton) 2019-01, Vol.58 (1), p.65-71
Main Authors: Kamerkar, Sukrut C, Roy, Krishnendu, Bhattacharyya, Soumya, Pucadyil, Thomas J
Format: Article
Language:English
Subjects:
Animals
Biological Transport
Brain - metabolism
Cell Membrane - chemistry
Cell Membrane - metabolism
Cytosol - metabolism
Dynamins - metabolism
Goats
Guanosine Triphosphate - metabolism
High-Throughput Screening Assays
Humans
Vesicular Transport Proteins - metabolism
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Summary:Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin. Biochemical fractionation followed by mass spectrometric analyses identifies the Eps15-homology domain-containing protein1 (EHD1) as a novel ATP-dependent membrane fission catalyst. Together, our approach establishes an experimental workflow for the discovery of novel membrane fission catalysts.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.8b00925