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

Abstract

BackgroundGiardia 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.MethodSequence 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.ResultGlRpn10 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.ConclusionGlRpn10 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0737-1) contains supplementary material, which is available to authorized users.

Multiple paralogues of α-SNAP in Giardia lamblia exhibit independent subcellular localization and redistribution during encystation and stress

“…4b , upper panel) [ 37 ]. Such subcellular redistribution of proteins, without any change at the level of transcription, has been previously observed for other giardial proteins, such as β’COP subunit, Rab11, YiP, heavy chain of clathrin, DRP, ESCP and Rpn10 [ 13 , 38 – 40 ]. Thus, many regulatory changes in this protist appear to be dependent on protein relocation, rather than synthesis of new proteins.…”

“…Immunofluorescence was performed in trophozoites, encysting trophozoites (time of encystation indicated in respective figures) and cysts as previously described [ 40 ]. Briefly, cells were harvested by chilling the culture tubes on ice, followed by centrifugation at 1000× g for 10 min.…”

“…cDNA preparation and real-time PCR was performed as previously described, with primers designed against unique regions of giardial α-SNAPs (Additional file 2 : Table S4 ) [ 35 , 40 ]. PCR condition was as follows: initial denaturation at 95 °C for 5 min, second denaturation at 95 °C for 30 s, and annealing for 20 s at 56, 64.5 or 65.8 °C for SNAP 17224 , SNAP 16521 and SNAP 10856 , respectively.…”

The distinctive flagellar proteome of Euglena gracilis illuminates the complexities of protistan flagella adaptation

“…Proteasomes are present in spermatozoa flagella, with roles in spermatogenesis and capacitation (the final maturation of released sperm for fertilization) (Kerns et al ., 2016). In Giardia lamblia proteasome subunits are found only in flagellar pores which precede flagellar assembly (Sinha et al ., 2015) and no proteasome subunits have been detected in the C . reinhardtii flagella, suggesting that proteins ubiquitinated within the flagellum are transported to the cytoplasm via IFT for degradation (Long et al ., 2015).…”

“…Proteasomes are present in spermatozoa flagella, with roles in spermatogenesis and capacitation (the final maturation of released sperm for fertilization) (Kerns et al, 2016). In Giardia lamblia proteasome subunits are found only in flagellar pores which precede flagellar assembly (Sinha et al, 2015) and no proteasome subunits have been detected in the New Phytologist (2021) 232: 1323-1336 www.newphytologist.com Ó 2021 The Authors New Phytologist Ó 2021 New Phytologist Foundation C. reinhardtii flagella, suggesting that proteins ubiquitinated within the flagellum are transported to the cytoplasm via IFT for degradation (Long et al, 2015). Hence detection of proteasomes in the fully assembled flagella of E. gracilis suggests distinct mechanisms for flagella initiation and maturation processes representing an alternate strategy of protein degradation to C. reinhardtii.…”