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VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.

Piterman R, Braunstein I, Isakov E, Ziv T, Navon A, Cohen S, Stanhill A - Mol. Biol. Cell (2014)

Bottom Line: We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association.Multiubiquitination events within the VWA domain can further regulate S5a association.Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.

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ATF4 is an S5a-dependent UPS substrate. (A) S5a kd and control cells were treated with Velcade where indicated (10 μM, 1 h) and subjected to HA, S5a, and PSMA1 (loading control) immunoblots. Quantification of the polyubiquitinated ATF4 proteasomal substrate is indicated to the right. (B) Control and S5a kd cells were evaluated toward ATF4 degradation rates by cyclohexamide treatment, and time course ATF4 content evaluation was performed by immunoblots to HA, S5a, and PSMD14. Quantification of the ATF4 content is indicated to the right, showing the delayed degradation of ATF4 in the S5a kd cells. (C) S5a kd and control cells were treated as in A and subjected to a PSMA1 IP. Proteasomal and ATF4 content were revealed by HA and PSMD14 immunoblot. (D) S5a kd and control cells were treated with Velcade and subjected to a PSMA1 IP. Proteasomal and ubiquitin content was revealed using the ubiquitin and PSMD14 immunoblots.
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Figure 2: ATF4 is an S5a-dependent UPS substrate. (A) S5a kd and control cells were treated with Velcade where indicated (10 μM, 1 h) and subjected to HA, S5a, and PSMA1 (loading control) immunoblots. Quantification of the polyubiquitinated ATF4 proteasomal substrate is indicated to the right. (B) Control and S5a kd cells were evaluated toward ATF4 degradation rates by cyclohexamide treatment, and time course ATF4 content evaluation was performed by immunoblots to HA, S5a, and PSMD14. Quantification of the ATF4 content is indicated to the right, showing the delayed degradation of ATF4 in the S5a kd cells. (C) S5a kd and control cells were treated as in A and subjected to a PSMA1 IP. Proteasomal and ATF4 content were revealed by HA and PSMD14 immunoblot. (D) S5a kd and control cells were treated with Velcade and subjected to a PSMA1 IP. Proteasomal and ubiquitin content was revealed using the ubiquitin and PSMD14 immunoblots.

Mentions: To address the role of S5a in polyubiquitinated substrate degradation, we took advantage of a previous report in yeast showing impaired degradation of Gcn4 in rpn10Δ strains (Mayor et al., 2005). ATF4 is the functional mammalian homologue of Gcn4 (Harding et al., 2000) and is also subject to ubiquitin-proteasome system (UPS)–dependent degradation (Lassot et al., 2001). We therefore tested whether ATF4 degradation is S5a regulated. To this end, we evaluated control and S5a kd cells expressing ATF4 for their ATF4 content under normal and proteasomal inhibitory conditions. As seen in Figure 2A, Velcade treatment enabled detection of the otherwise undetectable polyubiquitinated ATF4. In contrast, polyubiquitinated ATF4 could be detected in the S5a kd cells even without Velcade treatment, implying a functional role of S5a in efficient ATF4 degradation (Figure 2A, right, quantifications). The extended half-life of ATF4 in the S5a kd cells is revealed in cycloheximide experiments, in which the consequences of the slower rate of decay are seen even at the initiating time point (Figure 2B). To evaluate whether the impaired ATF4 degradation observed in the S5a kd cells is due to impaired proteasomal recruitment or inefficient proteasomal processing of the polyubiquitinated ATF4, we immunopurified proteasomes from both control and S5a kd cells and evaluated their ATF4 content. As observed in Figure 2C, in spite of higher ATF4 levels in the S5a kd cells (input), upon proteasome purification, reduced ATF4 levels are obtained in S5a kd cells, implying a recruitment role for S5a toward ATF4. The recruitment impairment was also observed when we monitored general proteasomal polyubiquitin content. We detected lower polyubiquitin levels upon proteasomal purifications in S5a kd cells in spite of their higher levels in the cell (Figure 2D, input vs. PSMA1 immunoprecipitation [IP]). This result implies a recruitment role for S5a toward polyubiquitinated substrates, as expected from a ubiquitin-binding proteasomal component.


VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.

Piterman R, Braunstein I, Isakov E, Ziv T, Navon A, Cohen S, Stanhill A - Mol. Biol. Cell (2014)

ATF4 is an S5a-dependent UPS substrate. (A) S5a kd and control cells were treated with Velcade where indicated (10 μM, 1 h) and subjected to HA, S5a, and PSMA1 (loading control) immunoblots. Quantification of the polyubiquitinated ATF4 proteasomal substrate is indicated to the right. (B) Control and S5a kd cells were evaluated toward ATF4 degradation rates by cyclohexamide treatment, and time course ATF4 content evaluation was performed by immunoblots to HA, S5a, and PSMD14. Quantification of the ATF4 content is indicated to the right, showing the delayed degradation of ATF4 in the S5a kd cells. (C) S5a kd and control cells were treated as in A and subjected to a PSMA1 IP. Proteasomal and ATF4 content were revealed by HA and PSMD14 immunoblot. (D) S5a kd and control cells were treated with Velcade and subjected to a PSMA1 IP. Proteasomal and ubiquitin content was revealed using the ubiquitin and PSMD14 immunoblots.
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Related In: Results  -  Collection

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Figure 2: ATF4 is an S5a-dependent UPS substrate. (A) S5a kd and control cells were treated with Velcade where indicated (10 μM, 1 h) and subjected to HA, S5a, and PSMA1 (loading control) immunoblots. Quantification of the polyubiquitinated ATF4 proteasomal substrate is indicated to the right. (B) Control and S5a kd cells were evaluated toward ATF4 degradation rates by cyclohexamide treatment, and time course ATF4 content evaluation was performed by immunoblots to HA, S5a, and PSMD14. Quantification of the ATF4 content is indicated to the right, showing the delayed degradation of ATF4 in the S5a kd cells. (C) S5a kd and control cells were treated as in A and subjected to a PSMA1 IP. Proteasomal and ATF4 content were revealed by HA and PSMD14 immunoblot. (D) S5a kd and control cells were treated with Velcade and subjected to a PSMA1 IP. Proteasomal and ubiquitin content was revealed using the ubiquitin and PSMD14 immunoblots.
Mentions: To address the role of S5a in polyubiquitinated substrate degradation, we took advantage of a previous report in yeast showing impaired degradation of Gcn4 in rpn10Δ strains (Mayor et al., 2005). ATF4 is the functional mammalian homologue of Gcn4 (Harding et al., 2000) and is also subject to ubiquitin-proteasome system (UPS)–dependent degradation (Lassot et al., 2001). We therefore tested whether ATF4 degradation is S5a regulated. To this end, we evaluated control and S5a kd cells expressing ATF4 for their ATF4 content under normal and proteasomal inhibitory conditions. As seen in Figure 2A, Velcade treatment enabled detection of the otherwise undetectable polyubiquitinated ATF4. In contrast, polyubiquitinated ATF4 could be detected in the S5a kd cells even without Velcade treatment, implying a functional role of S5a in efficient ATF4 degradation (Figure 2A, right, quantifications). The extended half-life of ATF4 in the S5a kd cells is revealed in cycloheximide experiments, in which the consequences of the slower rate of decay are seen even at the initiating time point (Figure 2B). To evaluate whether the impaired ATF4 degradation observed in the S5a kd cells is due to impaired proteasomal recruitment or inefficient proteasomal processing of the polyubiquitinated ATF4, we immunopurified proteasomes from both control and S5a kd cells and evaluated their ATF4 content. As observed in Figure 2C, in spite of higher ATF4 levels in the S5a kd cells (input), upon proteasome purification, reduced ATF4 levels are obtained in S5a kd cells, implying a recruitment role for S5a toward ATF4. The recruitment impairment was also observed when we monitored general proteasomal polyubiquitin content. We detected lower polyubiquitin levels upon proteasomal purifications in S5a kd cells in spite of their higher levels in the cell (Figure 2D, input vs. PSMA1 immunoprecipitation [IP]). This result implies a recruitment role for S5a toward polyubiquitinated substrates, as expected from a ubiquitin-binding proteasomal component.

Bottom Line: We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association.Multiubiquitination events within the VWA domain can further regulate S5a association.Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.

Show MeSH