Limits...
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

Functional complementation with GlRpn10. (a)S. cerevisiae rpn10∆ strain was transformed individually with each of the constructs expressing the proteins shown in Panel b. The growth of these transformed yeast cells was monitored by spot test using serial dilutions on YCM plates lacking uracil and containing galactose and canavanine. To ensure that equal number of cells have been used, spotting was also done on YCM plates lacking uracil and containing glucose. All the plates were incubated at 30 °C. (b) Schematic diagrams of GlRpn10, ScRpn10, and different deletion variants of these two proteins. The regions corresponding to the two domains, VWA and the UIM, are denoted in blue and green respectively. The K residues within the VWA domain of ScRpn10 are marked and their respective positions are indicated above. (c) Western blot using anti-ubiquitin antibody of the total cell extract of wild-type, rpn10∆ and rpn10∆ transformed with the above mentioned constructs. The composition of the growth medium is same as given in (a) above, except that these transformants were grown in liquid medium. Extracts were loaded in the following order: lane 1, Wild-type transformed with vector; lane 2, rpn10∆ transformed with vector; lane 3, rpn10∆ cells expressing GlRpn10; lane 4, rpn10∆ cells expressing ScRpn10; lane 5, rpn10∆ cells expressing ScRpn10*; lane 6, rpn10∆ cells expressing GlRpn10* and lane 7, rpn10∆ cells expressing GlRpn10•. 3-PGK was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Functional complementation with GlRpn10. (a)S. cerevisiae rpn10∆ strain was transformed individually with each of the constructs expressing the proteins shown in Panel b. The growth of these transformed yeast cells was monitored by spot test using serial dilutions on YCM plates lacking uracil and containing galactose and canavanine. To ensure that equal number of cells have been used, spotting was also done on YCM plates lacking uracil and containing glucose. All the plates were incubated at 30 °C. (b) Schematic diagrams of GlRpn10, ScRpn10, and different deletion variants of these two proteins. The regions corresponding to the two domains, VWA and the UIM, are denoted in blue and green respectively. The K residues within the VWA domain of ScRpn10 are marked and their respective positions are indicated above. (c) Western blot using anti-ubiquitin antibody of the total cell extract of wild-type, rpn10∆ and rpn10∆ transformed with the above mentioned constructs. The composition of the growth medium is same as given in (a) above, except that these transformants were grown in liquid medium. Extracts were loaded in the following order: lane 1, Wild-type transformed with vector; lane 2, rpn10∆ transformed with vector; lane 3, rpn10∆ cells expressing GlRpn10; lane 4, rpn10∆ cells expressing ScRpn10; lane 5, rpn10∆ cells expressing ScRpn10*; lane 6, rpn10∆ cells expressing GlRpn10* and lane 7, rpn10∆ cells expressing GlRpn10•. 3-PGK was used as loading control.

Mentions: RPN10 is a non-essential gene as the growth of yeast mutants with deletion of chromosomal RPN10 (rpn10Δ) is indistinguishable from that of wild-type cells at 30°C. However, when the cells are subjected to stress by growing them in the presence of amino acid analogues, such as canavanine (analogue of arginine), rpn10∆ cells fail to grow at 30°C [29]. This is because replacement of arginine with canavanine in the growth media results in production of defective proteins, which leads to increase in the misfolded protein load within the cell. Since this situation can only be countered with a fully functional proteasome, ScRpn10 becomes essential for survival in the presence of canavanine. For the functional complementation study, RPN10 was deleted from yeast genome and as expected, the mutant was unable to grow on YCM plates containing canavanine (Figure 3a). Growth of this mutant was restored to wild-type levels when ScRpn10 was expressed under the control of a galactose-inducible promoter (GAL1-10 promoter). Expression of GlRpn10 resulted in partial rescue of the growth phenotype of rpn10∆ cells (Figure 3a). This partial growth rescue phenotype of GlRpn10 may result from the absence of sequences from the N-terminal end of the GlRpn10 protein (Figure 1b) as a previous study has shown that a deletion of 61 amino acids from the N-terminus of ScRpn10 results in growth defects in the presence of amino acid analogues canavanine and p-flurophenylalanine [30]. The sequence alignment indicates that region of similarity between ScRpn10 and GlRpn10 starts around the 60th residue of the yeast protein (VLSTF sequence in ScRpn10) (Figure 1b). Using the present assay conditions, a deletion of the first 58 residues of ScRpn10 (ScRpn10*) also resulted in partial rescue of the growth phenotype of rpn10∆ and the extent of the partial rescue was similar to that observed with GlRpn10 (Figure 3a, compare GlRpn10 and ScRpn10*). Thus it may be concluded that the identified GlRpn10 protein is most likely to function as a component of the yeast proteasome. However, it is not fully functional as it lacks the N-terminal segment of the VWA domain.Figure 3


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)

Functional complementation with GlRpn10. (a)S. cerevisiae rpn10∆ strain was transformed individually with each of the constructs expressing the proteins shown in Panel b. The growth of these transformed yeast cells was monitored by spot test using serial dilutions on YCM plates lacking uracil and containing galactose and canavanine. To ensure that equal number of cells have been used, spotting was also done on YCM plates lacking uracil and containing glucose. All the plates were incubated at 30 °C. (b) Schematic diagrams of GlRpn10, ScRpn10, and different deletion variants of these two proteins. The regions corresponding to the two domains, VWA and the UIM, are denoted in blue and green respectively. The K residues within the VWA domain of ScRpn10 are marked and their respective positions are indicated above. (c) Western blot using anti-ubiquitin antibody of the total cell extract of wild-type, rpn10∆ and rpn10∆ transformed with the above mentioned constructs. The composition of the growth medium is same as given in (a) above, except that these transformants were grown in liquid medium. Extracts were loaded in the following order: lane 1, Wild-type transformed with vector; lane 2, rpn10∆ transformed with vector; lane 3, rpn10∆ cells expressing GlRpn10; lane 4, rpn10∆ cells expressing ScRpn10; lane 5, rpn10∆ cells expressing ScRpn10*; lane 6, rpn10∆ cells expressing GlRpn10* and lane 7, rpn10∆ cells expressing GlRpn10•. 3-PGK was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Functional complementation with GlRpn10. (a)S. cerevisiae rpn10∆ strain was transformed individually with each of the constructs expressing the proteins shown in Panel b. The growth of these transformed yeast cells was monitored by spot test using serial dilutions on YCM plates lacking uracil and containing galactose and canavanine. To ensure that equal number of cells have been used, spotting was also done on YCM plates lacking uracil and containing glucose. All the plates were incubated at 30 °C. (b) Schematic diagrams of GlRpn10, ScRpn10, and different deletion variants of these two proteins. The regions corresponding to the two domains, VWA and the UIM, are denoted in blue and green respectively. The K residues within the VWA domain of ScRpn10 are marked and their respective positions are indicated above. (c) Western blot using anti-ubiquitin antibody of the total cell extract of wild-type, rpn10∆ and rpn10∆ transformed with the above mentioned constructs. The composition of the growth medium is same as given in (a) above, except that these transformants were grown in liquid medium. Extracts were loaded in the following order: lane 1, Wild-type transformed with vector; lane 2, rpn10∆ transformed with vector; lane 3, rpn10∆ cells expressing GlRpn10; lane 4, rpn10∆ cells expressing ScRpn10; lane 5, rpn10∆ cells expressing ScRpn10*; lane 6, rpn10∆ cells expressing GlRpn10* and lane 7, rpn10∆ cells expressing GlRpn10•. 3-PGK was used as loading control.
Mentions: RPN10 is a non-essential gene as the growth of yeast mutants with deletion of chromosomal RPN10 (rpn10Δ) is indistinguishable from that of wild-type cells at 30°C. However, when the cells are subjected to stress by growing them in the presence of amino acid analogues, such as canavanine (analogue of arginine), rpn10∆ cells fail to grow at 30°C [29]. This is because replacement of arginine with canavanine in the growth media results in production of defective proteins, which leads to increase in the misfolded protein load within the cell. Since this situation can only be countered with a fully functional proteasome, ScRpn10 becomes essential for survival in the presence of canavanine. For the functional complementation study, RPN10 was deleted from yeast genome and as expected, the mutant was unable to grow on YCM plates containing canavanine (Figure 3a). Growth of this mutant was restored to wild-type levels when ScRpn10 was expressed under the control of a galactose-inducible promoter (GAL1-10 promoter). Expression of GlRpn10 resulted in partial rescue of the growth phenotype of rpn10∆ cells (Figure 3a). This partial growth rescue phenotype of GlRpn10 may result from the absence of sequences from the N-terminal end of the GlRpn10 protein (Figure 1b) as a previous study has shown that a deletion of 61 amino acids from the N-terminus of ScRpn10 results in growth defects in the presence of amino acid analogues canavanine and p-flurophenylalanine [30]. The sequence alignment indicates that region of similarity between ScRpn10 and GlRpn10 starts around the 60th residue of the yeast protein (VLSTF sequence in ScRpn10) (Figure 1b). Using the present assay conditions, a deletion of the first 58 residues of ScRpn10 (ScRpn10*) also resulted in partial rescue of the growth phenotype of rpn10∆ and the extent of the partial rescue was similar to that observed with GlRpn10 (Figure 3a, compare GlRpn10 and ScRpn10*). Thus it may be concluded that the identified GlRpn10 protein is most likely to function as a component of the yeast proteasome. However, it is not fully functional as it lacks the N-terminal segment of the VWA domain.Figure 3

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