Abstract
The yeast N-BAR (Bin/Amphiphysin/Rvs167) protein Rvs167 is recruited by the Rab GTPase Activating Proteins (RabGAP) Gyp5 and Gyl1 to the tip of small buds to act in exocytosis. Investigating other N-BAR proteins involved in Gyp5/Gyl1/Rvs167 complexes, we found that Rvs161, an Rvs167 paralog, is absent from the complexes formed at the tip of small buds. Immunoprecipitation and Bimolecular Fluorescence Complementation (BiFC) analysis show that both Rvs167 and Rvs161 interact in vivo with Gvp36, an N-BAR protein. Rvs167 molecules also interact independently of Rvs161 and Gvp36. Rvs167/Rvs167 and Rvs167/Gyp5 interactions predominate over other combinations at the tip of small buds, suggesting that N-BAR lattices enriched in Rvs167 molecules form at these sites. By combining BiFC with markers specific to each organelle, we analyzed systematically in living cells the locations of the BiFC signals generated by combinations of the three N-BAR proteins. We show that the BiFC signals differ according to organelle and cell site, strongly suggesting heterogeneity in the composition of N-BAR protein lattices in vivo. Our results reveal that the organization of N-BAR protein lattices in vivo is complex and are consistent with N-BAR proteins forming various types of dimers and lattices of variable composition.
Highlights
The BAR (Bin/Amphiphysin/Rvs167) domain superfamily is made up of highly conserved domains formed of dimeric alpha-helix coiled-coils
Gyp[5] acts as Rab GTPase Activating Proteins (RabGAP) protein towards Sec[419,24] and we have shown that Gyp[5] and Gyl[1] contribute to the regulation of exocytosis at the stage of polarized growth of the bud[21]
No significant defect was found either in rvs161Δ or gvp36Δ strains, at time-points when rvs167Δ cells displayed a significant decrease in Bgl2-HA secretion (Supplementary Fig. S2b). This indicates that, unlike Rvs[167], Rvs[161] and Gvp[36] are not involved in the control of secretion at the small-bud stage. These results indicate that the N-BAR lattices formed at the tip of small buds are enriched in Rvs[167] molecules, which interact with Gyp[5] and are involved in secretion at the small-bud stage independently of Rvs[161] and Gvp[36]
Summary
The BAR (Bin/Amphiphysin/Rvs167) domain superfamily is made up of highly conserved domains formed of dimeric alpha-helix coiled-coils. The BAR protein superfamily includes N-BAR proteins, characterized by an additional N-terminal amphipathic helix which inserts into one leaflet of the membrane and promotes membrane curvature and dimerization[3,4]. Two N-BAR domain proteins were initially identified: the Rvs[167] protein and its paralog Rvs1617,8. Yeast rvs mutants display numerous defects, including reduced viability upon starvation, sensitivity to high salt and cytotoxic compounds, defects in actin polarization, defects in endocytosis and random budding of diploid cells[10]. Rvs[167] and Rvs[161] can bind and tubulate membranes in vitro[11] One of their best understood functions is endocytosis: rvs161Δ and rvs167Δ are defective for α-factor internalization[8]. There has still been no demonstration of a physical interaction between Gvp[36] and either Rvs[167] or Rvs[161]
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