ABSTRACT
Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin.
Subject(s)
Actins/metabolism , Cell Membrane/metabolism , Peptide Elongation Factor 1/metabolism , Peptide Elongation Factors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , rho GTP-Binding Proteins/metabolism , Actins/genetics , Cell Membrane/genetics , Peptide Elongation Factor 1/genetics , Peptide Elongation Factors/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/genetics , Vacuoles/metabolism , rho GTP-Binding Proteins/geneticsABSTRACT
A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.