Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes

作者：Cai Bishuang; Giridharan Sai Srinivas Panapakkam; Zhang Jing; Saxena Sugandha; Bahl Kriti; Schmidt John A; Sorgen Paul L; Guo Wei; Naslavsky Naava; Caplan Steve^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2013, 288(42): 30172-30180.

DOI：10.1074/jbc.M113.488627

• Cell Biology
• Endosomes
• Liposomes
• Membrane Biogenesis
• Membrane Recycling
• Membrane Trafficking
• Protein Sorting
• Receptor Recycling
• Subcellular Organelles
• Vesicles

Background: Vesiculation of tubular recycling endosomes is essential for the recycling of receptors and lipids to the plasma membrane. Results: A novel vesiculation assay was used to demonstrate a role for endocytic regulatory proteins in vesiculation. Conclusion: EHD family proteins play significant roles in both the vesiculation and generation of tubular recycling endosomes. Significance: This provides the first direct evidence of differential EHD function in vesiculation and tubulation. %26lt;br%26gt;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.

• 出版日期2013-10-18