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Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

Nkosi PJ, Targosz BS, Labib K, Sanchez-Diaz A - PLoS ONE (2013)

Bottom Line: This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3.Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors.The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

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Physical interactions between Rvs167 and Inn1.(A) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (B) Scheme explaining how the indicated protein fragments were expressed in cultures of E. coli cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (C) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown E. coli protein is included to provide a loading control. (D) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.
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pone-0057846-g002: Physical interactions between Rvs167 and Inn1.(A) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (B) Scheme explaining how the indicated protein fragments were expressed in cultures of E. coli cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (C) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown E. coli protein is included to provide a loading control. (D) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.

Mentions: Hof1, Cyk3 and Iqg1 all interact with Inn1 [23], [24], [25], and we used the 2-hybrid assay to show that the same was true for Rvs167 (Figure 2A). The interaction was specific, as a range of other proteins with SH3 domains were unable to associate with Inn1 in the same assay (Figure S4A; [24]). The interaction required the function of the SH3 domain of Rvs167 (Figure S4B) but also involved the region rich in Glycine-Proline-Alanine that separates the BAR and SH3 domains of Rvs167 (Figure 2A). An equivalent fragment of Rvs167 co-purified specifically with the Proline-rich region of Inn1 from an extract of E. coli cells, whereas the SH3 domain alone did not (Figure 2B-D). This shows that the interaction of Rvs167 and Inn1 is direct and also explains why it was not detected in previous systematic studies of the targets of yeast SH3 proteins including Rvs167, as these studies focussed on the minimal SH3 domains [37], [38].


Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

Nkosi PJ, Targosz BS, Labib K, Sanchez-Diaz A - PLoS ONE (2013)

Physical interactions between Rvs167 and Inn1.(A) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (B) Scheme explaining how the indicated protein fragments were expressed in cultures of E. coli cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (C) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown E. coli protein is included to provide a loading control. (D) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.
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Related In: Results  -  Collection

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pone-0057846-g002: Physical interactions between Rvs167 and Inn1.(A) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (B) Scheme explaining how the indicated protein fragments were expressed in cultures of E. coli cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (C) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown E. coli protein is included to provide a loading control. (D) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of E. coli cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.
Mentions: Hof1, Cyk3 and Iqg1 all interact with Inn1 [23], [24], [25], and we used the 2-hybrid assay to show that the same was true for Rvs167 (Figure 2A). The interaction was specific, as a range of other proteins with SH3 domains were unable to associate with Inn1 in the same assay (Figure S4A; [24]). The interaction required the function of the SH3 domain of Rvs167 (Figure S4B) but also involved the region rich in Glycine-Proline-Alanine that separates the BAR and SH3 domains of Rvs167 (Figure 2A). An equivalent fragment of Rvs167 co-purified specifically with the Proline-rich region of Inn1 from an extract of E. coli cells, whereas the SH3 domain alone did not (Figure 2B-D). This shows that the interaction of Rvs167 and Inn1 is direct and also explains why it was not detected in previous systematic studies of the targets of yeast SH3 proteins including Rvs167, as these studies focussed on the minimal SH3 domains [37], [38].

Bottom Line: This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3.Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors.The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

View Article: PubMed Central - PubMed

Affiliation: Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

Show MeSH