Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.Plasmodium falciparum causes one of the most life-threatening infectious diseases of humans. Malaria is estimated to be responsible for up to 2 million deaths per year (34). The pathogenesis of the disease is associated with the intraerythrocytic cycle of the parasite, involving repeated rounds of invasion, growth, and schizogony. The erythrocyte provides a ready source of protein building blocks, but this quiescent cell provides little in the way of cellular architecture, as it possesses no internal organelles and no protein synthesis or trafficking machinery. As the parasite develops, it effectively remodels its adopted home by generating membranous structures outside its own cell and by implementing a complex and unusual system for transporting proteins across the host cell compartment and to its surface. This has led to particular interest in the membrane-bound compartments that appear in the red blood cell (RBC) cytoplasm as the parasite matures.Once the parasite has invaded a new host cell, it resides within a parasitophorous vacuole (PV). In the ring stage of intraerythrocytic growth, electron microscopy studies have revealed finger-like extensions of the PV membrane (5,11,43). These extensions are thought to remain connected to the PV and to develop to form a tubulovesicular network (TVN). As the parasite matures, disk-like structures appear at the RBC periphery, characterized by a translucent lumen and an electron-dense coat of variable thickness (4,11,22). These structures are referred to as Maurer's clefts, which is something of a misnomer, as they do not appear to be formed by invagina-

SummaryDuring the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.

The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a ␤(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors.The differentiation of Giardia lamblia cells to cysts is a key step in the simple life cycle of this ubiquitous intestinal parasite. Induced trophozoites, the flagellated, motile, and attachment-competent stage, exit the cell cycle at the G 2 -M transition (1) and begin to synthesize the components of an extracellular matrix, the cyst wall (CW). Synthesis and export of the cyst wall proteins (CWPs) to specialized organelles, termed encystation-specific vesicles (ESVs), is completed between 8 and 10 h postinduction (p.i.). After proteolytic cleavage of the CWP2 C terminus, the CWPs are sorted into two fractions which are deposited sequentially and polymerize on the surface of the cyst (18). The first layer of the CW, which eventually completely encloses the parasite (19,24), is secreted in the last minutes of differentiation simultaneously with nuclear division and morphological transformation of the cell (cyst formation). In vitro, the entire encystation process typically takes 20 to 24 h in our hands and can be induced by modifying medium components and/or the concentrations of bile, fatty acids (cholesterol), or lactic acid (3,10,15,22). Because the condition(s) which induce encystation in vivo are not known, all factors and components mentioned above are implicated since their concentrations vary in the small intestine, as does the pH, which increases from ϳ6 in the duodenum to 7.5 to 8.0 in the distal ileum. Although all available data indicate that differentiation is induced by one or several environmental signals, the mechanism(s) for reception and transduction have not been characterized. Upregulation...

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of `naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.