The Spir actin organizers are involved in vesicle transport processes

Abstract

The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.

Structural and functional insights into the Spir/formin actin nucleator complex

“…In somatic cells, the small G protein Rab11 is a master regulator of recycling and exocytic vesicle transport processes (Wang et al, 2008;Goldenring and Nam, 2011). The Spir proteins encode a modified FYVE zinc finger membrane-binding domain at the C-terminal end (Kerkhoff et al, 2001). FYVE zinc fingers are typically found in proteins involved in membrane trafficking (Hurley, 2006).…”

“…Early on after the discovery of Spir proteins, transient expression studies in NIH 3T3 fibroblast cells showed that the actin nucleators accumulate at vesicle membranes where they induce the accumulation of filamentous actin (Otto et al, 2000). The integrity of the FYVE zinc finger and the N-terminal adjacent Spir-box, which is a potential small G protein interaction site, were shown to mediate the distinct subcellular localization of the Spir proteins (Kerkhoff et al, 2001). Spir proteins were found to colocalize with the Rab11 protein in somatic cells.…”

“…A functional correlation of Rab11 and Spir resulted from overexpression experiments of a dominant interfering Spir mutant, which could localize to vesicle membranes, but lacked the N-terminal actin and formin-binding sequences (Spir-1-CT) (Kerkhoff et al, 2001). Transient Spir-1-CT expression in NIH 3T3 mouse fibroblasts and monkey Vero kidney epithelial cells blocked the exocytosis of the vesicular stomatitis virus G protein from the trans-Golgi apparatus to the plasma membrane, a process that was shown before to be regulated by Rab11 (Chen et al, 1998;Kerkhoff et al, 2001). In human HeLa cells, RNA interference knockdown experiments showed a function of Spir-1 in transport processes beyond early endosomes (Morel et al, 2009).…”

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

“…Besides a function in actin-dependent long range vesicle transport in oocytes, a role of Spir proteins in exocytic vesicle transport and in transport processes beyond early endosomes was described in somatic cells (7,8). In all cell types characterized so far, Spir proteins highly colocalize with Rab11, indicating a close functional relationship of the actin organizer and the small G protein of the Ras superfamily.…”

“…The localization of Spir proteins at intracellular membranes is mediated by a C-terminal FYVE-type zinc finger membrane interaction module, a structural domain that is typically found in proteins involved in membrane trafficking (7). FYVE zinc fingers comprise eight cysteine residues, which bind two zinc ions (20,21).…”