ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

Natalya Pashkova; Lokesh Gakhar; Liping Yu; Nicholas J Schnicker; Annabel Y Minard; Stanley Winistorfer; Ivan E Johnson; Robert C Piper

doi:10.7554/eLife.72583

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ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

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ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

Natalya Pashkova, Lokesh Gakhar, Liping Yu, Nicholas J Schnicker, Annabel Y Minard, Stanley Winistorfer, Ivan E Johnson and Robert C Piper

eLife, Vol.10, e72583

11/25/2021

DOI: 10.7554/eLife.72583

PMCID: PMC8648300

PMID: 34821552

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Abstract

Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here, we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

Mutation

Clathrin - metabolism

Endocytosis

Membrane Proteins - genetics

Membrane Proteins - metabolism

Protein Binding

Protein Domains

Saccharomyces cerevisiae - metabolism

Ubiquitin - metabolism

Vesicular Transport Proteins - metabolism

Details

Title: Subtitle: ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals
Creators: Natalya Pashkova - University of Iowa
Lokesh Gakhar - University of Iowa
Liping Yu - University of Iowa
Nicholas J Schnicker - Roy J. and Lucille A. Carver College of Medicine
Annabel Y Minard - University of Iowa
Stanley Winistorfer - University of Iowa
Ivan E Johnson - University of Iowa
Robert C Piper - University of Iowa
Resource Type: Journal article
Publication Details: eLife, Vol.10, e72583
DOI: 10.7554/eLife.72583
PMID: 34821552
PMCID: PMC8648300
NLM abbreviation: Elife
ISSN: 2050-084X
eISSN: 2050-084X
Grant note: R01 GM058202 / NIGMS NIH HHS
Language: English
Date published: 11/25/2021
Academic Unit: Molecular Physiology and Biophysics; Biochemistry and Molecular Biology; Medicine Administration; Internal Medicine
Record Identifier: 9984297596802771

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