title: A reduced VWA domain-containing proteasomal ubiquitin receptor of Giardia lamblia localizes to the flagellar pore regions in microtubule-dependent manner.
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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Sinha
authors.affiliation: Department of Biochemistry, Bose Institute, P 1/12, C. I. T. Road, Scheme - VII M, Kolkata, 700054, West Bengal, India. shankari@jcbose.ac.in.
authors.forename: Shankari Prasad
authors.initials: SP
authors.lastname: Datta
authors.affiliation: Department of Biochemistry, Bose Institute, P 1/12, C. I. T. Road, Scheme - VII M, Kolkata, 700054, West Bengal, India. atrayeeray@jcbose.ac.in.
authors.forename: Atrayee
authors.initials: A
authors.lastname: Ray
authors.affiliation: Department of Biochemistry, Bose Institute, P 1/12, C. I. T. Road, Scheme - VII M, Kolkata, 700054, West Bengal, India. srimonti@jcbose.ac.in.
authors.forename: Srimonti
authors.initials: S
authors.lastname: Sarkar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016829:Giardia lamblia / Q000235:genetics / Q000378:metabolism*; D005873:Giardiasis / Q000469:parasitology*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D053058:Trophozoites; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex