title: Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes.
abstract: Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Bishuang
authors.initials: B
authors.lastname: Cai
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Sai Srinivas Panapakkam
authors.initials: SSP
authors.lastname: Giridharan
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Sugandha
authors.initials: S
authors.lastname: Saxena
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Kriti
authors.initials: K
authors.lastname: Bahl
authors.affiliation: the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: John A
authors.initials: JA
authors.lastname: Schmidt
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Sorgen
authors.affiliation: the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and.
authors.forename: Naava
authors.initials: N
authors.lastname: Naslavsky
authors.affiliation: From the Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870 and. Electronic address: scaplan@unmc.edu.
authors.forename: Steve
authors.initials: S
authors.lastname: Caplan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D060588:LIM Domain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000096985:Calponins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D009687:Nuclear Proteins; D033921:Vesicular Transport Proteins; C120700:EHD1 protein, human; C406447:EHD3 protein, human; C406448:EHD4 protein, human; C455272:MICAL1 protein, human; D000096985:Calponins
keywords: Cell Biology; Endosomes; Liposomes; Membrane Biogenesis; Membrane Recycling; Membrane Trafficking; Protein Sorting; Receptor Recycling; Subcellular Organelles; Vesicles
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