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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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rsp5 and bre5 mutations have additive effect on growth and mislocalization of GFP-Rer1.(A) bre5Δ and HA-rsp5-1 mutations show weak genetic interaction. Strains doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 were transformed with plasmid encoding DOA4. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (B) doa4Δ bre5Δ HA-rsp5-1 mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.
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pone-0039582-g001: rsp5 and bre5 mutations have additive effect on growth and mislocalization of GFP-Rer1.(A) bre5Δ and HA-rsp5-1 mutations show weak genetic interaction. Strains doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 were transformed with plasmid encoding DOA4. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (B) doa4Δ bre5Δ HA-rsp5-1 mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.

Mentions: It has been reported that the double rsp5 ubp3Δ mutant is inviable at a temperature permissive for the single rsp5 and ubp3Δ mutants and the rsp5 and ubp3Δ mutations have an additive effect on ribophagy [20]. Additionally, Ubp3p, together with its cofactor Bre5p, is involved in deubiquitination of Sec23p, a subunit of COPII coat. Recently it was shown that Sec23p is ubiquitinated by Rsp5p [21]. The single ubp3Δ and bre5Δ mutants were shown to accumulate ubiquitinated Sec27p, a β’-COPI subunit [22]. Therefore, we asked if Rsp5p also acts with the Ubp3p-Bre5p complex in retrograde Golgi-to-ER trafficking. First we tested the genetic interaction between rsp5-1 mutation, which carries an amino acid substitution in the catalytic Hect domain, and deletion of the BRE5 gene (bre5Δ) in a doa4Δ background (see below for explanation). A comparison of growth of strains doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 on YPD at different temperatures revealed that the doa4Δ bre5Δ HA-rsp5-1 mutant grew worse at 35°C than did the doa4Δ bre5Δ or doa4Δ HA-rsp5-1 mutants, indicating a very weak genetic interaction between bre5Δ and rsp5-1 (not shown). The same genetic interaction was visible when all above strains were transformed with a plasmid bearing the DOA4 gene (Figure 1A). This interaction is in agreement with data from a genetic interaction map [23].


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

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

rsp5 and bre5 mutations have additive effect on growth and mislocalization of GFP-Rer1.(A) bre5Δ and HA-rsp5-1 mutations show weak genetic interaction. Strains doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 were transformed with plasmid encoding DOA4. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (B) doa4Δ bre5Δ HA-rsp5-1 mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383674&req=5

pone-0039582-g001: rsp5 and bre5 mutations have additive effect on growth and mislocalization of GFP-Rer1.(A) bre5Δ and HA-rsp5-1 mutations show weak genetic interaction. Strains doa4Δ, doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 were transformed with plasmid encoding DOA4. Serial 1∶10 dilutions of transformants were spotted on YPD and incubated at indicated temperatures. (B) doa4Δ bre5Δ HA-rsp5-1 mutant accumulates GFP-Rer1 in vacuole. Plasmid encoding GFP-Rer1 fusion was transformed into same mutants as in panel A. Transformants were grown on SC -ura at 28°C and GFP-Rer1 was observed by fluorescence (GFP). Cells were stained with CMAC to visualize vacuole. Percentage of cells accumulating GFP in vacuole is given.
Mentions: It has been reported that the double rsp5 ubp3Δ mutant is inviable at a temperature permissive for the single rsp5 and ubp3Δ mutants and the rsp5 and ubp3Δ mutations have an additive effect on ribophagy [20]. Additionally, Ubp3p, together with its cofactor Bre5p, is involved in deubiquitination of Sec23p, a subunit of COPII coat. Recently it was shown that Sec23p is ubiquitinated by Rsp5p [21]. The single ubp3Δ and bre5Δ mutants were shown to accumulate ubiquitinated Sec27p, a β’-COPI subunit [22]. Therefore, we asked if Rsp5p also acts with the Ubp3p-Bre5p complex in retrograde Golgi-to-ER trafficking. First we tested the genetic interaction between rsp5-1 mutation, which carries an amino acid substitution in the catalytic Hect domain, and deletion of the BRE5 gene (bre5Δ) in a doa4Δ background (see below for explanation). A comparison of growth of strains doa4Δ bre5Δ, doa4Δ HA-rsp5-1 and doa4Δ bre5Δ HA-rsp5-1 on YPD at different temperatures revealed that the doa4Δ bre5Δ HA-rsp5-1 mutant grew worse at 35°C than did the doa4Δ bre5Δ or doa4Δ HA-rsp5-1 mutants, indicating a very weak genetic interaction between bre5Δ and rsp5-1 (not shown). The same genetic interaction was visible when all above strains were transformed with a plasmid bearing the DOA4 gene (Figure 1A). This interaction is in agreement with data from a genetic interaction map [23].

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