Over-Expression of Rififylin, a New RING Finger and FYVE-like Domain-containing Protein, Inhibits Recycling from the Endocytic Recycling Compartment

Abstract

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

Endocytic Recycling Compartments Altered in Cisplatin-Resistant Cancer Cells

“…The overall distribution of the ERC in these cells is related to the amount and distribution of stable detyrosinated microtubules. The structural and functional integrity of the ERC in part depends on the cytoskeleton, which seems to be involved in regulating molecular sorting and vesicular transport of the ERC with microtubules (36). In this work, we found that B104-5 cells sensitive to temperature are more resistant to cisplatin, with an altered ERC due to stable Glu-a-tubulin-containing microtubules, at nonpermissive temperature than at permissive temperature.…”

Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans

“…Exit of internalized transferrin out of the ERC has been commonly used as an assay of ERC function [33-37]. Indeed, the first evidence for the role of EHD1 in mammalian endocytic transport employed the ability of EHD1 G429R mutant to delay transferrin exit from the ERC [11].…”

“…While paradoxical, this finding is not unprecedented, as overexpression of other proteins involved in endocytic traffic, such as Rififylin, the RING finger and FYVE-like domain ERC protein, also led to a block in transferrin recycling [33] perhaps due to disruption of functional protein complexes and/or sequestration of effector proteins. Overexpression of EHD1, 3 and 4 ΔEH mutants further increased the transferrin exit block while that of EHD2 ΔEH did not (Figure 9B, grey).…”

Three-Dimensional Structure of the Human Cytomegalovirus Cytoplasmic Virion Assembly Complex Includes a Reoriented Secretory Apparatus

“…Early endosomes are not monolithic entities; rather, they come in tubular and vesicular forms and vary in the complement of associated proteins. In some cells, EEA1 is a more specific marker for early endosomes than is rab5 (17), but EEA1-negative/rab5-positive early endosomes have been identified (7). The structure of the cytoplasmic inclusion in naturally infected renal tubule cells (15) is at least superficially similar to its structure in infected cultured fibroblasts.…”