Protein trafficking in Giardia lamblia

Giardia, a protozoan parasite of humans and other vertebrates, is a common cause of intestinal disease worldwide. Besides its medical importance, Giardia is considered an excellent system to study the evolution of fundamental cellular processes because it belongs to the earliest branches of the eukaryotic lineage of descent. Giardia trophozoites lack organelles typical of higher eukaryotes such mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, such as the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important human pathogen.     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

Human and Giardia ADP-ribosylation factors (ARFs) complement ARF function in Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. ARFs are highly conserved, ubiquitously expressed in eukaryotic cells and appear to be involved in vesicular protein transport. The two yeast ARFs are > 60% identical to mammalian ARFs and are essential for cell viability (Stearns, T., Kahn, R. A., Botstein, D., and Hoyt, M. A. (1990) Mol. Cell. Biol. 10, 6690-6699). Although the two yeast ARF proteins are 96% identical in amino acid sequence, the yeast ARF1 gene is constitutively expressed, whereas the ARF2 gene is repressed by glucose. Human ARF5 and ARF6 and a Giardia ARF differ substantially in size and amino acid identity from other mammalian and eukaryotic ARFs but will, as befits their designation, activate cholera toxin. Expression of human ARF5, ARF6, or Giardia ARF cDNA rescued the lethal yeast ARF double mutant (arf1, arf2). Strains rescued by human ARF5, ARF6, or Giardia ARF grew much more slowly than wild-type yeast or strains rescued with yeast ARF1. We infer from the impaired growth of these rescued strains that the homologous ARFs may have specific targeting information that does not interact effectively or efficiently with the yeast protein membrane trafficking system.     

Emergent properties of proteostasis-COPII coupled systems in human health and disease

In eukaryotic membrane trafficking, emergent protein folding pathways dictated by the proteostasis network (the 'PN') in each cell type are linked to the coat protein complex II (COPII) system that initiates transport through the exocytic pathway. These coupled pathways direct the transit of protein cargo from the endoplasmic reticulum (ER) to diverse subcellular and extracellular destinations. Understanding how the COPII system selectively manages the trafficking of distinct folded states of nascent cargo (comprising one-third of the proteins synthesized by the eukaryotic genome) in close cooperation with the PN remains a formidable challenge to the field. Whereas the PN may contain a thousand component, the minimal COPII coat components that drive all vesicle budding from the ER include Sar1 (a GTPase), Sec12 (a guanine nucleotide exchange factor), Sec23-Sec24 complexes (protein cargo selectors) and the Sec13-Sec31 complex (that functions as a protein cargo collector and as a polymeric lattice generator to promote vesicle budding). A wealth of data suggests a hierarchical role of the PN and COPII components in coupling protein folding with recruitment and assembly of vesicle coats on the ER. In this minireview, we focus on insights recently gained from the study of inherited human disease states of the COPII machinery. We explore the relevance of the COPII system to human biology in the context of its inherent link with the remarkably flexible folding capacity of the PN in each cell type and in response to the environment. The pharmacological manipulation of this coupled system has important therapeutic implications for restoration of function in human disease.     

Split introns in the genome of Giardia intestinalis are excised by spliceosome-mediated trans-splicing

Spliceosomal introns are hallmarks of most eukaryotic genomes and are excised from premature mRNAs by a spliceosome that is among the largest, and most complex, molecular machine in cells. The divergent unicellular eukaryote Giardia intestinalis, the causative agent of giardiasis, also possesses spliceosomes, but only four canonical (cis-spliced) introns have been identified in its genome to date. We demonstrate that this organism has a novel form of spliceosome-mediated trans-splicing of split introns that is essential for generating mature mRNAs for at least two important genes: one encoding a heat shock protein 90 (HSP90), which controls the conformation of a suite of cellular proteins, and the other encoding a dynein molecular motor protein, involved in the motility of eukaryotic flagella. These split introns have properties that distinguish them from other trans-splicing systems known within eukaryotes, suggesting that Giardia independently evolved a unique system to splice split introns.     

Organelles of protein transport in Giardia lamblia

Giardia occupies a unique evolutionary position since it is considered to belong to the earliest known lineage to diverge from the eukaryotic line of descent. Although organelles of protein transport are thought to have evolved with the nuclear membrane, G. lamblia is reported to have no Golgi apparatus. Therefore, Frances Gillin, David Reiner and Michael McCaffery have investigated how it exports glycoproteins to the cyst wall during encystation and whether a Golgi might become evident during an active secretory phase. They have found both functional and morphological evidence of a Golgi in Giardia and have shown that trophozoites are capable of sophisticated protein recognition, sorting and trafficking. These studies suggest that membranous organelles of protein transport appeared early in the evolution of the eukaryotic cell.     

The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant-negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant-negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.     

GLUT4 trafficking in insulin-sensitive cells

In recent years, there have been major advances in the under-standing of both the cell biology of vesicle trafficking between intracellular compartments and the molecular targeting signals intrinsic to the trafficking proteins themselves. One system to which these advances have been profitably applied is the regulation of the trafficking of a glucose transporter, GLUT4, from intracellular compartment(s) to the cell surface in response to insulin. The unique nature of the trafficking of GLUT4 and its expression in highly differentiated cells makes this a question of considerable interest to cell biologists. Unraveling the tangled web of molecular events coordinating GLUT4 trafficking will eventually lead to a greater understanding of mammalian glucose metabolism, as well as fundamental cell biological principles related to organelle biogenesis and protein trafficking.     

Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia

Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa). Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.     

Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism

ABSTRACT: BACKGROUND: Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. RESULTS: Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. CONCLUSION: Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.     

Predation and eukaryote cell origins: a coevolutionary perspective

Cells are of only two kinds: bacteria, with DNA segregated by surface membrane motors, dating back approximately 3.5Gy; and eukaryotes, which evolved from bacteria, possibly as recently as 800-850My ago. The last common ancestor of eukaryotes was a sexual phagotrophic protozoan with mitochondria, one or two centrioles and cilia. Conversion of bacteria (=prokaryotes) into a eukaryote involved approximately 60 major innovations. Numerous contradictory ideas about eukaryogenesis fail to explain fundamental features of eukaryotic cell biology or conflict with phylogeny. Data are best explained by the intracellular coevolutionary theory, with three basic tenets: (1) the eukaryotic cytoskeleton and endomembrane system originated through cooperatively enabling the evolution of phagotrophy; (2) phagocytosis internalised DNA-membrane attachments, unavoidably disrupting bacterial division; recovery entailed the evolution of the nucleus and mitotic cycle; (3) the symbiogenetic origin of mitochondria immediately followed the perfection of phagotrophy and intracellular digestion, contributing greater energy efficiency and group II introns as precursors of spliceosomal introns. Eukaryotes plus their archaebacterial sisters form the clade neomura, which evolved from a radically modified derivative of an actinobacterial posibacterium that had replaced the ancestral eubacterial murein peptidoglycan by N-linked glycoproteins, radically modified its DNA-handling enzymes, and evolved cotranslational protein secretion, but not the isoprenoid-ether lipids of archaebacteria. I focus on this phylogenetic background and on explaining how in response to novel phagotrophic selective pressures and ensuing genome internalisation this prekaryote evolved efficient digestion of prey proteins by retrotranslocation and 26S proteasomes, then internal digestion by phagocytosis, lysosomes, and peroxisomes, and eukaryotic vesicle trafficking and intracellular compartmentation.     

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.     

Giardia lamblia -- a model organism for eukaryotic cell differentiation

Giardia lamblia is a binucleated, flagellated protozoan parasite that inhabits the upper small intestine of its vertebrate hosts. The entire life cycle, which can be completed in vitro, is simple with cycling between a vegetative trophozoite and a highly resistant cystic form. The parasite is one of the earliest diverging eukaryotes known and more than 95% of the genome is sequenced. This makes Giardia an excellent model system for studies of basic eukaryotic processes like cell differentiation. In this review we will discuss recent data concerning Giardia differentiation with a focus on DNA replication and cytokinesis.     

Membrane trafficking pathways in Alzheimer's disease

Membrane proteins are constantly being trafficked in cells and the relevant proteins in Alzheimer's disease (AD), such as the amyloid precursor protein (APP) and its processing enzymes, are not exempted from that. Molecular cell biologists have been endeavoring to ascertain a roadmap for APP processing and trafficking in various cell types including neurons. This has led to the identification of numerous regulatory sorting mechanisms, protein-protein interactions and lipidic microenvironments that largely define how and where the substrate APP meets its processing enzymes. However, the cell biology of tau, and the formation of neurofibrillary tangles, has long been regarded as a separate field. Nonetheless, recent progress is bringing both worlds together in a new paradigm on how Aβ toxicity and tau are physiologically connected. Here, we discuss an update of our current appraisal on how membrane trafficking may play an important role in the pathogenesis of the disease and how this could be exploited for effective therapy.     

The lipid-droplet proteome reveals that droplets are a protein-storage depot

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.     

The Legionella pneumophila Effector VipA Is an Actin Nucleator That Alters Host Cell Organelle Trafficking

Legionella pneumophila, the causative agent of Legionnaires'' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.     

The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins.

We have investigated the use of Leishmania cells as a novel eukaryotic expression system for the production of recombinant protein. These cells are easy to maintain, requiring no CO2 incubator or shaker, and can be grown in standard tissue culture media. Leishmania cells can be readily transfected with plasmid DNA by electroporation and transformants selected with antibiotic resistance. Recent studies have shown that it is possible to express foreign genes in Leishmania for the purpose of understanding the biology of this protozoan cell. In the present study we report the use of this system as a means of producing a biologically functional human p53 protein. The conformation of the p53 protein is critical for its ability to bind specific DNA sequences. It is demonstrated that Leishmania-synthesized human p53 is phosphorylated and can bind specifically to its enhancer DNA sequence. These data demonstrate that Leishmania may represent a simple eukaryotic expression system for the production of biologically active recombinant proteins.     

Protein prenylation: a new mode of host-pathogen interaction

Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction.     

Large-scale profiling of Rab GTPase trafficking networks: the membrome

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.