Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface.

Combined biochemical and cytological analysis of membrane trafficking using lectins.

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans.

Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs).

The genome of the African trypanosome Trypanosoma brucei.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T.

An evolutionarily conserved coiled-coil protein implicated in polycystic kidney disease is involved in basal body duplication and flagellar biogenesis in Trypanosoma brucei.

Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease.

Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite.

To investigate the role of clathrin-mediated trafficking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the beta-adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three beta-adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and beta-adaptin1 are constitutively expressed throughout the Leishmania life cycle.

New approaches to the microscopic imaging of Trypanosoma brucei.

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 x 10(7) disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 x 10(6) DALYs.

The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis.

Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP.

Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers.

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum.

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system.

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed.

The kinetoplastida endocytic apparatus. Part I: a dynamic system for nutrition and evasion of host defences.

The endocytic system of kinetoplastid parasites is a highly polarized membrane network focused on the flagellar pocket localized at one end of the cell. When first characterized, the endosomal network was envisioned as a simple system for uptake of extracellular material by fluid-phase or receptor-mediated mechanisms. Subsequently, it has become clear that the kinetoplastid endosomal system has an active and vital role in avoiding the host immune system and virulence, as well as providing the basic functions to fulfil cellular nutritional requirements.

TbRAB18, a developmentally regulated Golgi GTPase from Trypanosoma brucei.

The trypanosomal secretory system is broadly similar to that of higher eukaryotes as proteins enter the system via the endoplasmic reticulum and are transported to the Golgi complex for elaboration of glycan chains. Importantly N-glycan processing is stage specific with only the bloodstream form (BSF) processing beyond the oligomannose form. Increased complexity of the BSF Golgi apparatus, as evidenced by morphological studies, may underpin this higher activity, but few trypanosome-specific Golgi proteins have been described that may play a role in this developmental alteration.