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Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

Jarmoszewicz K, Łukasiak K, Riezman H, Kaminska J - PLoS ONE (2012)

Bottom Line: We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate.Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits.Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

ABSTRACT
Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

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Retrograde trafficking from the Golgi to the ER is impaired in double arp2-1 rsp5-1 and arp2-1 ret1-1 mutants.(A-C) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (A) Defect of GFP-Rer1 trafficking in arp2-1 rsp5-1 mutants. Spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (B) Double mutant arp2-1 rsp5-1 is not sensitive to neomycin. Spore clones were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (C) Secretion of Kar2 is enhanced in arp2-1 rsp5-1 mutant. Spore clones were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting Kar2 were identified by Western blotting with anti-Kar2 antibody. (D–F) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (D) Defect of GFP-Rer1 trafficking in arp2-1 × ret1-1 mutant. The spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (E) Double mutant arp2-1 ret1-1 is sensitive to neomycin. The sensitivity to neomycin was tested as in B. (F) Secretion of Kar2p is enhanced in arp2-1 ret1-1 mutant. The secretion was assayed as in C.
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pone-0039582-g007: Retrograde trafficking from the Golgi to the ER is impaired in double arp2-1 rsp5-1 and arp2-1 ret1-1 mutants.(A-C) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (A) Defect of GFP-Rer1 trafficking in arp2-1 rsp5-1 mutants. Spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (B) Double mutant arp2-1 rsp5-1 is not sensitive to neomycin. Spore clones were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (C) Secretion of Kar2 is enhanced in arp2-1 rsp5-1 mutant. Spore clones were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting Kar2 were identified by Western blotting with anti-Kar2 antibody. (D–F) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (D) Defect of GFP-Rer1 trafficking in arp2-1 × ret1-1 mutant. The spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (E) Double mutant arp2-1 ret1-1 is sensitive to neomycin. The sensitivity to neomycin was tested as in B. (F) Secretion of Kar2p is enhanced in arp2-1 ret1-1 mutant. The secretion was assayed as in C.

Mentions: Several types of actin and actin-related proteins are found on Golgi membranes, including the GTPase Cdc42p which modulates actin cytoskeleton formation via the actin nucleating complex Arp2/3 interacts with Sec21p (γCOP) [3]. The rsp5 mutations show a genetic interaction with the arp2-1 mutation (ARP2 encodes a subunit of the Arp2/3 complex), and with mutations in the PAN1 gene or with deletions of LAS17 (PAN1 and LAS17 encode Arp2/3 complex activators) [17], [32], [33]. Therefore, we asked the question if Rsp5p acts in retrograde trafficking indirectly by influencing formation of the actin cytoskeleton. To test this hypothesis we first monitored the trafficking from Golgi-to-ER in arp2-1 mutant. As shown in Figure 7Aarp2-1 mutant alone does not have defect in trafficking of GFP-Rer1, is not sensitive to neomycin and does not secrete Kar2p as rsp5-1 cells do. The effect of both arp2-1 and rsp5-19 was also monitored. As shown in Figure 7A in double arp2-1 rsp5-19 mutant the GFP-Rer1 fusion was accumulated in vacuole. We did not observe an enhanced sensitivity to neomycin compared to wild type or to single mutants (Figure 7B), but Kar2p was secreted in the double mutant (Figure 7C). To further support the hypothesis that Rsp5p may influence Golgi-to-ER trafficking by regulating actin cytoskeleton organization we also tested genetic interaction between arp2-1 and ret1-1. The double arp2-1 ret1-1 mutant exhibited the same phenotypes as ret1-1 rsp5-1. It accumulated GFP-Rer1 in the vacuole (Figure 7D), was more sensitive to neomycin (Figure 7E) and secreted Kar2p (Figure 7F). Together these results shows that Arp2p and Rsp5p are important for the transport form the Golgi to the ER and support the hypothesis that Rsp5p influences trafficking from Golgi-to-ER indirectly by regulation of actin cytoskeleton dynamics.


Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

Jarmoszewicz K, Łukasiak K, Riezman H, Kaminska J - PLoS ONE (2012)

Retrograde trafficking from the Golgi to the ER is impaired in double arp2-1 rsp5-1 and arp2-1 ret1-1 mutants.(A-C) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (A) Defect of GFP-Rer1 trafficking in arp2-1 rsp5-1 mutants. Spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (B) Double mutant arp2-1 rsp5-1 is not sensitive to neomycin. Spore clones were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (C) Secretion of Kar2 is enhanced in arp2-1 rsp5-1 mutant. Spore clones were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting Kar2 were identified by Western blotting with anti-Kar2 antibody. (D–F) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (D) Defect of GFP-Rer1 trafficking in arp2-1 × ret1-1 mutant. The spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (E) Double mutant arp2-1 ret1-1 is sensitive to neomycin. The sensitivity to neomycin was tested as in B. (F) Secretion of Kar2p is enhanced in arp2-1 ret1-1 mutant. The secretion was assayed as in C.
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Related In: Results  -  Collection

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pone-0039582-g007: Retrograde trafficking from the Golgi to the ER is impaired in double arp2-1 rsp5-1 and arp2-1 ret1-1 mutants.(A-C) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (A) Defect of GFP-Rer1 trafficking in arp2-1 rsp5-1 mutants. Spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (B) Double mutant arp2-1 rsp5-1 is not sensitive to neomycin. Spore clones were serially diluted 1∶10, spotted on YPD or YPD containing 1 mM neomycin (Neo) and grown for 1 day at 28°C. (C) Secretion of Kar2 is enhanced in arp2-1 rsp5-1 mutant. Spore clones were replica-plated onto nitrocellulose filters and grown on solid YPD for 1 day at 28°C. Cells secreting Kar2 were identified by Western blotting with anti-Kar2 antibody. (D–F) Analysis of transport from the Golgi to ER in spore clones from cross arp2-1 × ret1-1. (D) Defect of GFP-Rer1 trafficking in arp2-1 × ret1-1 mutant. The spore clones were transformed with plasmid encoding GFP-RER1. Transformants were grown and analyzed similarly as spore clones in Figure 4B. (E) Double mutant arp2-1 ret1-1 is sensitive to neomycin. The sensitivity to neomycin was tested as in B. (F) Secretion of Kar2p is enhanced in arp2-1 ret1-1 mutant. The secretion was assayed as in C.
Mentions: Several types of actin and actin-related proteins are found on Golgi membranes, including the GTPase Cdc42p which modulates actin cytoskeleton formation via the actin nucleating complex Arp2/3 interacts with Sec21p (γCOP) [3]. The rsp5 mutations show a genetic interaction with the arp2-1 mutation (ARP2 encodes a subunit of the Arp2/3 complex), and with mutations in the PAN1 gene or with deletions of LAS17 (PAN1 and LAS17 encode Arp2/3 complex activators) [17], [32], [33]. Therefore, we asked the question if Rsp5p acts in retrograde trafficking indirectly by influencing formation of the actin cytoskeleton. To test this hypothesis we first monitored the trafficking from Golgi-to-ER in arp2-1 mutant. As shown in Figure 7Aarp2-1 mutant alone does not have defect in trafficking of GFP-Rer1, is not sensitive to neomycin and does not secrete Kar2p as rsp5-1 cells do. The effect of both arp2-1 and rsp5-19 was also monitored. As shown in Figure 7A in double arp2-1 rsp5-19 mutant the GFP-Rer1 fusion was accumulated in vacuole. We did not observe an enhanced sensitivity to neomycin compared to wild type or to single mutants (Figure 7B), but Kar2p was secreted in the double mutant (Figure 7C). To further support the hypothesis that Rsp5p may influence Golgi-to-ER trafficking by regulating actin cytoskeleton organization we also tested genetic interaction between arp2-1 and ret1-1. The double arp2-1 ret1-1 mutant exhibited the same phenotypes as ret1-1 rsp5-1. It accumulated GFP-Rer1 in the vacuole (Figure 7D), was more sensitive to neomycin (Figure 7E) and secreted Kar2p (Figure 7F). Together these results shows that Arp2p and Rsp5p are important for the transport form the Golgi to the ER and support the hypothesis that Rsp5p influences trafficking from Golgi-to-ER indirectly by regulation of actin cytoskeleton dynamics.

Bottom Line: We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate.Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits.Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

ABSTRACT
Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

Show MeSH
Related in: MedlinePlus