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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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S5a–hPlic interaction in the presence of proteasomes. (A) Soluble proteasomes were acquired from WT cells by performing PSMA1 IP, followed by a peptide elution. Cell lysates depleted from proteasomes were acquired by ultracentrifugation, precipitating the HMW 26S particle. Both sources of S5a (proteasomal and free) were directly evaluated with regard to their S5a, PSMA1, and hPlic content (input) or subjected to hPlic IP. Although proteasome fractions had only small quantities of hPlic (hPlic IP, long exposure), they did reveal the presence of S5a in the hPlic IP. Asterisk, light chain, immunoglobulin G (IgG). (B) WT or S5a-overexpressing cells were evaluated for their S5a distribution. Note the high S5a content in the LMW fractions upon S5a overexpression. Proteasome free lysates obtained from untransfected (un-Tx.) and transfected S5a cells (Tx. S5a) were subjected to hPLic IP, and in spite of the abundant S5a content in the proteasome-free fraction, S5a content was not revealed in the hPlic IP. Asterisk, light chain, IgG.
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Figure 4: S5a–hPlic interaction in the presence of proteasomes. (A) Soluble proteasomes were acquired from WT cells by performing PSMA1 IP, followed by a peptide elution. Cell lysates depleted from proteasomes were acquired by ultracentrifugation, precipitating the HMW 26S particle. Both sources of S5a (proteasomal and free) were directly evaluated with regard to their S5a, PSMA1, and hPlic content (input) or subjected to hPlic IP. Although proteasome fractions had only small quantities of hPlic (hPlic IP, long exposure), they did reveal the presence of S5a in the hPlic IP. Asterisk, light chain, immunoglobulin G (IgG). (B) WT or S5a-overexpressing cells were evaluated for their S5a distribution. Note the high S5a content in the LMW fractions upon S5a overexpression. Proteasome free lysates obtained from untransfected (un-Tx.) and transfected S5a cells (Tx. S5a) were subjected to hPLic IP, and in spite of the abundant S5a content in the proteasome-free fraction, S5a content was not revealed in the hPlic IP. Asterisk, light chain, IgG.

Mentions: Our data thus far indicate that Ubl proteasomal recruitment is mediated by S5a (Figure 3). To evaluate whether preferential binding of hPlic to proteasomal S5a exists, we examined whether the S5a-hPlic interaction that is observed on proteasomes (Figure 3B) can also occur in the absence of proteasomes. In spite of the low levels of hPlic in the proteasomal fractions (Figures 3A and 4A, long hPlic exposure), we were able to affinity purify hPlic from soluble proteasomes and detected S5a presence in this copurification (Figure 4A). However, no interaction between hPlic and S5a was detected in the absence of proteasomes, in spite of the high abundance of hPlic in these fractions (Figure 4A). To confirm that the lack of S5a binding to hPlic in the absence of proteasomes is not a due to the low quantities of S5a in these fractions (Figures 1C and 4B), we overexpressed S5a to increase its abundance in the lower–molecular weight (LMW) fractions. In spite of the observed increase (Figure 4B), we were unable to detect S5a presence upon hPlic immunoprecipitation (Figure 4B). We therefore conclude that S5a interaction with hPlic occurs only on the proteasome.


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)

S5a–hPlic interaction in the presence of proteasomes. (A) Soluble proteasomes were acquired from WT cells by performing PSMA1 IP, followed by a peptide elution. Cell lysates depleted from proteasomes were acquired by ultracentrifugation, precipitating the HMW 26S particle. Both sources of S5a (proteasomal and free) were directly evaluated with regard to their S5a, PSMA1, and hPlic content (input) or subjected to hPlic IP. Although proteasome fractions had only small quantities of hPlic (hPlic IP, long exposure), they did reveal the presence of S5a in the hPlic IP. Asterisk, light chain, immunoglobulin G (IgG). (B) WT or S5a-overexpressing cells were evaluated for their S5a distribution. Note the high S5a content in the LMW fractions upon S5a overexpression. Proteasome free lysates obtained from untransfected (un-Tx.) and transfected S5a cells (Tx. S5a) were subjected to hPLic IP, and in spite of the abundant S5a content in the proteasome-free fraction, S5a content was not revealed in the hPlic IP. Asterisk, light chain, IgG.
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Related In: Results  -  Collection

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Figure 4: S5a–hPlic interaction in the presence of proteasomes. (A) Soluble proteasomes were acquired from WT cells by performing PSMA1 IP, followed by a peptide elution. Cell lysates depleted from proteasomes were acquired by ultracentrifugation, precipitating the HMW 26S particle. Both sources of S5a (proteasomal and free) were directly evaluated with regard to their S5a, PSMA1, and hPlic content (input) or subjected to hPlic IP. Although proteasome fractions had only small quantities of hPlic (hPlic IP, long exposure), they did reveal the presence of S5a in the hPlic IP. Asterisk, light chain, immunoglobulin G (IgG). (B) WT or S5a-overexpressing cells were evaluated for their S5a distribution. Note the high S5a content in the LMW fractions upon S5a overexpression. Proteasome free lysates obtained from untransfected (un-Tx.) and transfected S5a cells (Tx. S5a) were subjected to hPLic IP, and in spite of the abundant S5a content in the proteasome-free fraction, S5a content was not revealed in the hPlic IP. Asterisk, light chain, IgG.
Mentions: Our data thus far indicate that Ubl proteasomal recruitment is mediated by S5a (Figure 3). To evaluate whether preferential binding of hPlic to proteasomal S5a exists, we examined whether the S5a-hPlic interaction that is observed on proteasomes (Figure 3B) can also occur in the absence of proteasomes. In spite of the low levels of hPlic in the proteasomal fractions (Figures 3A and 4A, long hPlic exposure), we were able to affinity purify hPlic from soluble proteasomes and detected S5a presence in this copurification (Figure 4A). However, no interaction between hPlic and S5a was detected in the absence of proteasomes, in spite of the high abundance of hPlic in these fractions (Figure 4A). To confirm that the lack of S5a binding to hPlic in the absence of proteasomes is not a due to the low quantities of S5a in these fractions (Figures 1C and 4B), we overexpressed S5a to increase its abundance in the lower–molecular weight (LMW) fractions. In spite of the observed increase (Figure 4B), we were unable to detect S5a presence upon hPlic immunoprecipitation (Figure 4B). We therefore conclude that S5a interaction with hPlic occurs only on the proteasome.

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
Related in: MedlinePlus