Srimonti Sarkar

Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase

A direct role for phosphoinositides in vesicular trafficking has been demonstrated by the identification of the yeast VPS34 gene encoding the phosphatidylinositol 3-kinase responsible for the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P). Vps34p binds the protein kinase Vps15p, and it has recently been shown that Vps15p and Vps34p associate with Vps30p and Vps38p to form a multimeric complex, termed complex II. We observed that mutations in the VPS30 and VPS38 genes led to a selective sorting and maturation phenotype of the soluble vacuolar protease CPY. Localization studies revealed that the CPY receptor Vps10p and the Golgi-endoprotease Kex2p were mislocalized to vacuolar membranes in strains deficient for either Vps30p or Vps38p, respectively. Interestingly, we measured decreased PtdIns3P levels in Δvps30 andΔ vps38 cells and observed redistribution of Vps5p and Vps17p to the cytoplasm in these mutants. Vps5p and Vps17p are subunits of the retromer complex that is required for endosome-to-Golgi retrograde transport. Both proteins contain the Phox homology (PX) domain, a recently identified phosphoinositide-binding motif. We demonstrate that the PX domains of Vps5p and Vps17p specifically bind to PtdIns3P in vitro and in vivo. On the basis of these and other observations, we propose that the PtdIns 3-kinase complex II directs the synthesis of a specific endosomal pool of PtdIns3P, which is required for recruitment/activation of the retromer complex, thereby ensuring efficient endosome-to-Golgi retrograde transport.

Defects in tRNA processing and nuclear export induce GCN4 translation independently of phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2.

ABSTRACT Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNAMetbinding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal (tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded byPUS4 also leads to translational derepression ofGCN4 (Gcd− phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd− phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate thatPUS4 overexpression did not diminish functional initiator tRNAMet levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd− phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyr that cannot be processed by RNase P had a Gcd− phenotype. Interestingly, the Gcd− phenotype of hcPUS4also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd− phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal* showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMet binding to the ribosome.

Comparative genomics reveals selective distribution and domain organization of FYVE and PX domain proteins across eukaryotic lineages

BackgroundPhosphatidylinositol 3-phosphate is involved in regulation of several key cellular processes, mainly endocytosis, signaling, nuclear processes, cytoskeletal remodelling, cell survival, membrane trafficking, phagosome maturation and autophagy. In most cases effector proteins bind to this lipid, using either FYVE or PX domain. These two domains are distributed amongst varied life forms such as virus, protists, fungi, viridiplantae and metazoa. As the binding ligand is identical for both domains, the goal of this study was to understand if there is any selectivity for either of these domains in different taxa. Further, to understand the different cellular functions that these domains may be involved in, we analyzed the taxonomic distribution of additional domains that associate with FYVE and PX.ResultsThere is selectivity for either FYVE or PX in individual genomes where both domains are present. Fungi and metazoa encode more PX, whereas streptophytes in viridiplantae encode more FYVE. Excess of FYVE in streptophytes results from proteins containing RCC1and DZC domains and FYVE domains in these proteins have a non-canonical ligand-binding site. Within a taxonomic group the selected domain associates with a higher number of other domains and is thus expected to discharge a larger number of cellular functions. Also, while certain associated domains are present in all taxonomic groups, most of them are unique to a specific group indicating that while certain common functions are discharged by these domains in all taxonomic groups, some functions appear to be group specific.ConclusionsAlthough both FYVE and PX bind to PtdIns(3)P, genomes of different taxa show distinct selectivity of encoding either of the two. Higher numbers of taxonomic group specific domains co-occur with the more abundant domain (FYVE/PX) indicating that group-specific rare domain architectures might have emerged to accomplish certain group-specific functions.

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

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.

Identification and Characterization of a FYVE Domain from the Early Diverging Eukaryote Giardia lamblia

The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

Significantly Diverged Did2/Vps46 Orthologues from the Protozoan Parasite Giardia lamblia

The endosomal compartment performs extensive sorting functions in most eukaryotes, some of which are accomplished with the help of the multivesicular body (MVB) sorting pathway. This pathway depends on the sequential action of complexes, termed the endosomal sorting complex required for transport (ESCRT). After successful sorting, the crucial step of recycling of the ESCRT complex components requires the activation of the AAA ATPase Vps4, and Did2/Vps46 plays an important role in this activation event. The endolysosomal system of the protozoan parasite Giardia lamblia appears to lack complexity, for instead of having distinct early endosomes, late endosomes and lysosomes, there are only peripheral vesicles (PVs) that are located close to the cell periphery. Additionally, comparative genomics studies predict the presence of only a subset of the ESCRT components in G. lamblia. Thus, it is possible that the MVB pathway is not functional in G. lamblia. To address this issue, the present study focused on the two putative orthologues of Did2/Vps46 of G. lamblia as their function is likely to be pivotal for a functional MVB sorting pathway. In spite of considerable sequence divergence, compared to other eukaryotic orthologues, the proteins encoded by both these genes have the ability to function as Did2/Vps46 in the context of the yeast ESCRT pathway. Furthermore, they also localized to the cellular periphery, where PVs are also located. Thus, this report is the first to provide experimental evidence indicating the presence of a functional ESCRT component in G. lamblia by characterizing the putative Did2/Vps46 orthologues.

Phosphoinositide binding profiles of the PX domains of Giardia lamblia

The membrane trafficking machinery that functions at the endomembrane system of Giardia lamblia appears to be significantly different from that present in most model eukaryotes. This machinery is important for encystation as cyst wall material is trafficked to the cell surface via encystation-specific vesicles. Since proteins containing the phosphoinositide-binding PX domains are known regulators of vesicular trafficking, BLAST search was used to identify the PX domains of G. lamblia. Six putative PX domain-containing ORFs were identified. Some of the encoded PX domains contained non-canonical amino acid residues in the highly conserved ligand binding pocket. In vitro and in vivo binding studies indicate that these domains have the ability to bind to diverse phosphoinositides. Also, coincidence detection is likely to play a significant role in ligand binding in vivo since domains that bind to the same lipid in vitro, exhibit differences in subcellular localization. Analyses of the expression of these six genes in trophozoites, encysting trophozoites and cysts showed that while the expression of four of the genes were downregulated in cysts, the other two were upregulated. The variation in ligand preference of the individual PX domains and the differential expression of most of the PX-domain encoding genes indicate that these PX domain-containing proteins are likely to perform diverse cellular functions.

The minimal ESCRT machinery of Giardia lamblia has altered inter-subunit interactions within the ESCRT-II and ESCRT-III complexes

The ESCRT pathway functions at different subcellular membranes to induce their negative curvature, and it has been largely characterized in model eukaryotes belonging to Opisthokonta. But searches of the genomes of many nonopisthokonts belonging to various supergroups indicate that some of them may harbour fewer ESCRT components. Of the genomes explored thus far, one of the most minimal set of ESCRT components was identified in the human pathogen Giardia lamblia, which belongs to Excavata. Here we report that an ESCRT-mediated pathway most likely operates at the peripheral vesicles, which are located at the cell periphery and the bare zone of this protist. Functional comparison of all the identified putative giardial ESCRT components, with the corresponding well-characterized orthologues from Saccharomyces cerevisiae, indicated that only some of the ESCRT components could functionally substitute for the corresponding yeast proteins. While GlVps25, GlVps2, and all three paralogues of GlVps4, tested positive in functional complementation assays, GlVps22, GlVps20, and GlVps24 did not. Binary interactions of either GlVps22 or GlVps25, with other ESCRT-II components from Giardia or yeast indicate that the giardial Vps36 orthologue is either completely missing or highly diverged. Interactions within the giardial ESCRT-III components also differ from those in yeast; while GlVps46a interacts preferentially with Vps24 compared to Vps2, GlVps46b, like the yeast orthologue, interacts with both.

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

BackgroundThe differently-diverged parasitic protist Giardia lamblia is known to have minimal machinery for vesicular transport. Yet, it has three paralogues of SNAP, a crucial component that together with NSF brings about disassembly of the cis-SNARE complex formed following vesicle fusion to target membranes. Given that most opisthokont hosts of this gut parasite express only one α-SNAP, this study was undertaken to determine whether these giardial SNAP proteins have undergone functional divergence.ResultsAll three SNAP paralogues are expressed in trophozoites, encysting trophozoites and cysts. Even though one of them clusters with γ-SNAP sequences in a phylogenetic tree, functional complementation analysis in yeast indicates that all the three proteins are functionally orthologous to α-SNAP. Localization studies showed a mostly non-overlapping distribution of these α-SNAPs in trophozoites, encysting cells and cysts. In addition, two of the paralogues exhibit substantial subcellular redistribution during encystation, which was also seen following exposure to oxidative stress. However, the expression of the three genes remained unchanged during this redistribution process. There is also a difference in the affinity of each of these α-SNAP paralogues for GlNSF.ConclusionsNone of the genes encoding the three α-SNAPs are pseudogenes and the encoded proteins are likely to discharge non-redundant functions in the different morphological states of G. lamblia. Based on the difference in the interaction of individual α-SNAPs with GlNSF and their non-overlapping pattern of subcellular redistribution during encystation and under stress conditions, it may be concluded that the three giardial α-SNAP paralogues have undergone functional divergence. Presence of one of the giardial α-SNAPs at the PDRs of flagella, where neither GlNSF nor any of the SNAREs localize, indicates that this α-SNAP discharges a SNARE-independent role in this gut pathogen.

The proteasome of the differently-diverged eukaryote Giardia lamblia and its role in remodeling of the microtubule-based cytoskeleton.

Abstract Giardia lamblia is the causative agent of the diarrheal disease giardiasis, against which only a limited number of drugs are currently available. Increasing reports of resistance to these drugs makes it necessary to identify new cellular targets for designing the next generation of anti-giardial drugs. Towards this goal, therapeutic agents that target the parasitic cellular machinery involved in the functioning of the unique microtubule-based cytoskeleton of the Giardia trophozoites are likely to be effective as microtubule function is not only important for the survival of trophozoites within the host, but also their extensive remodeling is necessary during the transition from trophozoites to cysts. Thus, drugs that affect microtubule remodeling have the potential to not only kill the disease-causing trophozoites, but also inhibit transmission of cysts in the community. Recent studies in other model organisms have indicated that the proteasome plays an integral role in the formation and remodeling of the microtubule-based cytoskeleton. This review draws attention to the various processes by which the giardial proteasome may impact the functioning of its microtubule cytoskeleton and highlights the possible differences of the parasitic proteasome and some of other cellular machinery involved in microtubule remodeling, compared to that of the higher eukaryotic host.