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A reduced VWA domain-containing proteasomal ubiquitin receptor of Giardia lamblia localizes to the flagellar pore regions in microtubule-dependent manner.

Sinha A, Datta SP, Ray A, Sarkar S - Parasit Vectors (2015)

Bottom Line: Besides the expected nuclear and cytosolic distribution, the protein displays microtubule-dependent flagellar pore localization in trophozoites.While the protein remained in the nucleus and cytosol in encysting trophozoites, it could no longer be detected at the flagellar pores.This absence at the flagellar pore regions in encysting trophozoites is likely to involve redistribution of the protein, rather than decreased gene expression or selective protein degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bose Institute, P 1/12, C. I. T. Road, Scheme - VII M, Kolkata, 700054, West Bengal, India. abhishek.boseinst.84@gmail.com.

ABSTRACT

Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the proteasome plays a pivotal role in this switching event. Compared to most model eukaryotes, the proteasome of this parasite has already been documented to have certain variations. This study was undertaken to characterize the ubiquitin receptor, GlRpn10, of the 19S regulatory particle of the Giardia proteasome and determine its cellular localization in trophozoites, encysting trophozoites and cysts.

Method: Sequence alignment and domain architecture analyses were performed to characterize GlRpn10. In vitro ubiquitin binding assay, functional complementation and biochemical studies verified the protein's ability to function as ubiquitin receptor in the context of the yeast proteasome. Immunofluorescence localization was performed with antibody against GlRpn10 to determine its distribution in trophozoites, encysting trophozoites and cysts. Real-time PCR and Western blotting were performed to monitor the expression pattern of GlRpn10 during encystation.

Result: GlRpn10 contained a functional ubiquitin interacting motif, which was capable of binding to ubiquitin. Although it contained a truncated VWA domain, it was still capable of partially complementing the function of the yeast Rpn10 orthologue. Apart from localizing to the nucleus and cytosol, GlRpn10 was also present at flagellar pores of trophozoites and this localization was microtubule-dependent. Although there was no change in the cellular levels of GlRpn10 during encystation, its selective distribution at the flagellar pores was absent.

Conclusion: GlRpn10 contains a noncanonical VWA domain that is partially functional in yeast. Besides the expected nuclear and cytosolic distribution, the protein displays microtubule-dependent flagellar pore localization in trophozoites. While the protein remained in the nucleus and cytosol in encysting trophozoites, it could no longer be detected at the flagellar pores. This absence at the flagellar pore regions in encysting trophozoites is likely to involve redistribution of the protein, rather than decreased gene expression or selective protein degradation.

No MeSH data available.


Related in: MedlinePlus

In vitroubiquitin binding assay of UIM of GlRpn10 subunit. UIM of GlRpn10 and ScVps27 were tagged with 6XHis tag. The isolated proteins were immobilized on Ni-NTA agarose beads and allowed to bind GST-tagged ubiquitin. Following elution with imidazole, the eluate was analyzed by Western blotting with anti-GST antibody. GlUIM* represents S → E mutant protein of GlRpn10 UIM. One-fifth of the eluate volume used for the anit-GST blot, was run on a separate gel and stained with coomassie blue.
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Fig2: In vitroubiquitin binding assay of UIM of GlRpn10 subunit. UIM of GlRpn10 and ScVps27 were tagged with 6XHis tag. The isolated proteins were immobilized on Ni-NTA agarose beads and allowed to bind GST-tagged ubiquitin. Following elution with imidazole, the eluate was analyzed by Western blotting with anti-GST antibody. GlUIM* represents S → E mutant protein of GlRpn10 UIM. One-fifth of the eluate volume used for the anit-GST blot, was run on a separate gel and stained with coomassie blue.

Mentions: A recent study has led to the identification of some of the components of the Giardia proteasome by performing tandem affinity purification with the tagged Rpt1 orthologue, followed by mass spectrometry [7]. This resulted in the identification of a putative GlRpn10, which is encoded by the ORF GL50803_15604. However, no orthologue of the other ubiquitin receptor, Rpn13, was identified. BLAST searches of the Giardia genome with the Rpn13 orthologues from various eukaryotes also failed to identify any putative orthologue of this protein (AS and SS, unpublished results). Even the putative GlRpn10 protein shared very low sequence identity (16.8%) with the S. cerevisiae Rpn10 (ScRpn10), thus raising concerns about its capability to function as an ubiquitin receptor of the proteasome. With the aim of functionally characterizing the putative GlRpn10 orthologue, domain architecture analysis of the protein sequence was performed using Pfam and multiple sequence alignment was done to compare the sequence of putative GlRpn10 with sequences of Rpn10 orthologues derived from various eukaryotes like A. mellifera, H. sapiens, S. cerevisiae, M. crystallinum, and C. parvum (Figure 1a and b). The Rpn10 protein is known to contain two different domains, a VWA domain located towards the N-terminus, and one or more UIMs located after the VWA (Figure 1a). There is variability in the UIM repeat number; while the S. cerevisiae orthologue has a single UIM, the human orthologue has two and the fly orthologues (Drosophila and Apis) have three (Figure 1a and b) [8]. Analysis of the predicted amino acid sequence of GlRpn10 in Pfam indicates that it contains only a single UIM and no other domain (Figure 1a). The predicted UIM of GlRpn10 contains all conserved residues that are characteristic of UIMs. This include the N-terminal acidic amino acids (EDDIE), followed by a large hydrophobic residue (L), an A present two amino acid away, followed by a conserved S at 13th position of the domain (Figure 1b) [26]. This S is crucial for the recognition of ubiquitin and change of this residue to negatively charged amino acid (D or E) hampers the ubiquitin recognition property of UIM [22]. It is known that the UIM domains of S5a (human homolog of Rpn10 that contains two UIMs) have varying affinity for ubiquitin; the UIM of S5a located more towards the N-terminus, has lower affinity for ubiquitin compared to the UIM located after it [27,9]. The UIM of GlRpn10 aligns with the N-terminal UIM of S5a (Figure 1b) rather than the next UIM. Thus, it is possible that the UIM of GlRpn10 may have low affinity for ubiquitin. To test this, the ability of GlRpn10 to bind with ubiquitin was determined by performing in vitro ubiquitin-binding assay wherein binding between UIM and GST-ubiquitin (GST-Ub) was ascertained. For this purpose UIM of GlRpn10 was tagged with a 6xHis tag. As positive control, the 6xHis-tagged UIM derived from S. cerevisiae Vps27 was used and an unrelated domain of Giardia, FYVE, served as negative control [22,23]. While selective retention of GST-Ub was observed for UIM domains derived from Vps27 and GlRpn10, no retention was evident for the 6xHis-tagged FYVE domain (Figure 2, lanes 1, 2 and 4). Lack of binding between Vps27 UIM and GST alone, or between the Ni-NTA resin and GST-Ub served as additional negative controls for this in vitro assay (Figure 2, lanes 5 and 6).Figure 1


A reduced VWA domain-containing proteasomal ubiquitin receptor of Giardia lamblia localizes to the flagellar pore regions in microtubule-dependent manner.

Sinha A, Datta SP, Ray A, Sarkar S - Parasit Vectors (2015)

In vitroubiquitin binding assay of UIM of GlRpn10 subunit. UIM of GlRpn10 and ScVps27 were tagged with 6XHis tag. The isolated proteins were immobilized on Ni-NTA agarose beads and allowed to bind GST-tagged ubiquitin. Following elution with imidazole, the eluate was analyzed by Western blotting with anti-GST antibody. GlUIM* represents S → E mutant protein of GlRpn10 UIM. One-fifth of the eluate volume used for the anit-GST blot, was run on a separate gel and stained with coomassie blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352536&req=5

Fig2: In vitroubiquitin binding assay of UIM of GlRpn10 subunit. UIM of GlRpn10 and ScVps27 were tagged with 6XHis tag. The isolated proteins were immobilized on Ni-NTA agarose beads and allowed to bind GST-tagged ubiquitin. Following elution with imidazole, the eluate was analyzed by Western blotting with anti-GST antibody. GlUIM* represents S → E mutant protein of GlRpn10 UIM. One-fifth of the eluate volume used for the anit-GST blot, was run on a separate gel and stained with coomassie blue.
Mentions: A recent study has led to the identification of some of the components of the Giardia proteasome by performing tandem affinity purification with the tagged Rpt1 orthologue, followed by mass spectrometry [7]. This resulted in the identification of a putative GlRpn10, which is encoded by the ORF GL50803_15604. However, no orthologue of the other ubiquitin receptor, Rpn13, was identified. BLAST searches of the Giardia genome with the Rpn13 orthologues from various eukaryotes also failed to identify any putative orthologue of this protein (AS and SS, unpublished results). Even the putative GlRpn10 protein shared very low sequence identity (16.8%) with the S. cerevisiae Rpn10 (ScRpn10), thus raising concerns about its capability to function as an ubiquitin receptor of the proteasome. With the aim of functionally characterizing the putative GlRpn10 orthologue, domain architecture analysis of the protein sequence was performed using Pfam and multiple sequence alignment was done to compare the sequence of putative GlRpn10 with sequences of Rpn10 orthologues derived from various eukaryotes like A. mellifera, H. sapiens, S. cerevisiae, M. crystallinum, and C. parvum (Figure 1a and b). The Rpn10 protein is known to contain two different domains, a VWA domain located towards the N-terminus, and one or more UIMs located after the VWA (Figure 1a). There is variability in the UIM repeat number; while the S. cerevisiae orthologue has a single UIM, the human orthologue has two and the fly orthologues (Drosophila and Apis) have three (Figure 1a and b) [8]. Analysis of the predicted amino acid sequence of GlRpn10 in Pfam indicates that it contains only a single UIM and no other domain (Figure 1a). The predicted UIM of GlRpn10 contains all conserved residues that are characteristic of UIMs. This include the N-terminal acidic amino acids (EDDIE), followed by a large hydrophobic residue (L), an A present two amino acid away, followed by a conserved S at 13th position of the domain (Figure 1b) [26]. This S is crucial for the recognition of ubiquitin and change of this residue to negatively charged amino acid (D or E) hampers the ubiquitin recognition property of UIM [22]. It is known that the UIM domains of S5a (human homolog of Rpn10 that contains two UIMs) have varying affinity for ubiquitin; the UIM of S5a located more towards the N-terminus, has lower affinity for ubiquitin compared to the UIM located after it [27,9]. The UIM of GlRpn10 aligns with the N-terminal UIM of S5a (Figure 1b) rather than the next UIM. Thus, it is possible that the UIM of GlRpn10 may have low affinity for ubiquitin. To test this, the ability of GlRpn10 to bind with ubiquitin was determined by performing in vitro ubiquitin-binding assay wherein binding between UIM and GST-ubiquitin (GST-Ub) was ascertained. For this purpose UIM of GlRpn10 was tagged with a 6xHis tag. As positive control, the 6xHis-tagged UIM derived from S. cerevisiae Vps27 was used and an unrelated domain of Giardia, FYVE, served as negative control [22,23]. While selective retention of GST-Ub was observed for UIM domains derived from Vps27 and GlRpn10, no retention was evident for the 6xHis-tagged FYVE domain (Figure 2, lanes 1, 2 and 4). Lack of binding between Vps27 UIM and GST alone, or between the Ni-NTA resin and GST-Ub served as additional negative controls for this in vitro assay (Figure 2, lanes 5 and 6).Figure 1

Bottom Line: Besides the expected nuclear and cytosolic distribution, the protein displays microtubule-dependent flagellar pore localization in trophozoites.While the protein remained in the nucleus and cytosol in encysting trophozoites, it could no longer be detected at the flagellar pores.This absence at the flagellar pore regions in encysting trophozoites is likely to involve redistribution of the protein, rather than decreased gene expression or selective protein degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bose Institute, P 1/12, C. I. T. Road, Scheme - VII M, Kolkata, 700054, West Bengal, India. abhishek.boseinst.84@gmail.com.

ABSTRACT

Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the proteasome plays a pivotal role in this switching event. Compared to most model eukaryotes, the proteasome of this parasite has already been documented to have certain variations. This study was undertaken to characterize the ubiquitin receptor, GlRpn10, of the 19S regulatory particle of the Giardia proteasome and determine its cellular localization in trophozoites, encysting trophozoites and cysts.

Method: Sequence alignment and domain architecture analyses were performed to characterize GlRpn10. In vitro ubiquitin binding assay, functional complementation and biochemical studies verified the protein's ability to function as ubiquitin receptor in the context of the yeast proteasome. Immunofluorescence localization was performed with antibody against GlRpn10 to determine its distribution in trophozoites, encysting trophozoites and cysts. Real-time PCR and Western blotting were performed to monitor the expression pattern of GlRpn10 during encystation.

Result: GlRpn10 contained a functional ubiquitin interacting motif, which was capable of binding to ubiquitin. Although it contained a truncated VWA domain, it was still capable of partially complementing the function of the yeast Rpn10 orthologue. Apart from localizing to the nucleus and cytosol, GlRpn10 was also present at flagellar pores of trophozoites and this localization was microtubule-dependent. Although there was no change in the cellular levels of GlRpn10 during encystation, its selective distribution at the flagellar pores was absent.

Conclusion: GlRpn10 contains a noncanonical VWA domain that is partially functional in yeast. Besides the expected nuclear and cytosolic distribution, the protein displays microtubule-dependent flagellar pore localization in trophozoites. While the protein remained in the nucleus and cytosol in encysting trophozoites, it could no longer be detected at the flagellar pores. This absence at the flagellar pore regions in encysting trophozoites is likely to involve redistribution of the protein, rather than decreased gene expression or selective protein degradation.

No MeSH data available.


Related in: MedlinePlus