Secretory traffic in the eukaryotic parasite Toxoplasma gondii: less is more

Abstract

Name a single-celled eukaryote that boasts a small genome size, is easily cultivated in haploid form, for which a wide variety of molecular genetic tools are available, and that exhibits a simple, polarized secretory apparatus with a well-defined endoplasmic reticulum and Golgi that can serve as a model for understanding secretion. Got it? Now name a cell with all these attributes that contains at least a dozen distinct and morphologically well-defined intracellular organelles, including three distinct types of secretory vesicles and two endosymbiotic organelles. Not so sure anymore?

Figure 1.

Intracellular parasitophorous vacuole containing two T. gondii parasites within a human host cell. The ER is distributed throughout the cell, but predominantly in the basal region. The Golgi apparatus is invariably found adjacent to the apical end of the nucleus. Rhoptries and micronemes are found at the apical end of the parasite (terminating in the conoid region), whereas dense granules are distributed throughout the cell. The inner membrane complex is comprised of a series of closed sacs of uncertain origin, underlying the plasma membrane. The micropore (not visible in this micrograph) is the only stable structure bridging the parasite plasma membrane and inner membrane complex. Clathrin-coated vesicles are often observed at the micropore (Nichols et al., 1994), suggesting that endocytosis may occur at this site. Bar, 2 μm. Ap, Apicoplast; Co, conoid; DG, dense granule; ER, endoplasmic reticulum; Go, Golgi; IMC, inner membrane complex; Mn, microneme; Mt, mitochondrion; Nu, nucleus; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; Rh, rhoptry; HC, host cell cytoplasm; HC-ER, host cell endoplasmic reticulum; HC-Mt; host cell mitochondrion.

Figure 2.

Fluorescent protein labeling of subcellular organelles in T. gondii . Fusions between endogenous parasite proteins and GFP, YFP, or other reporters have been expressed in transgenic T. gondii, and localization has been determined by fluorescence microscopy. The central cartoon, showing subcellular structures (also see Fig. 1), illustrates proper targeting of GFP chimeras. Labeling of the conoid and subpellicular microtubules was achieved using a YFP–α-tubulin construct (Striepen et al., 2000; Hu et al., 2002; Swedlow et al., 2002), micronemes using MIC3-GFP (Striepen et al., 2001), Golgi using MIC3[68–137]-GFP (Striepen et al., 2001), mitochondria using HSP60-GFP (Hu et al., 2001), plasma membrane using P30-GFP-GPI (Striepen et al., 2000); rhoptries using ROP1-GFP (Striepen et al., 1998), dense granules using P30-GFP (Striepen et al., 1998), nucleus using PCNA-GFP (Radke et al., 2001), ER using P30-GFP-HDEL (Hager et al., 1999), and inner membrane complex using IMC1-YFP (Hu et al., 2001).

Figure 3.

Post-Golgi protein targeting in the T. gondii secretory pathway. Protein traffic through the ER and Golgi likely depends on both COPI- and COPII-coated vesicles, and is regulated by forward targeting signals, ER retrieval and retention motifs, and Rab proteins. Targeting of soluble proteins from the trans-Golgi network to dense granules is signal independent, whereas targeting of membrane proteins to these organelles depends on transmembrane domain length (unpublished data). T. gondii rab6 mediates retrograde transport from dense granules to the parasite Golgi (unpublished data). Rhoptry proteins appear likely to be transported from the Golgi via a precursor compartment, possibly part of the endosomal pathway (Robibaro et al., 2002). Transmembrane rhoptry proteins are targeted in a tyrosine-, dileucine-, and adaptor-dependent fashion. Targeting of soluble microneme proteins proceeds by association with transmembrane escorters; transmembrane proteins are capable of using adaptor- and tyrosine-dependent signals, although typical endocytic motifs are not apparent in known microneme proteins. Results using dominant–negative adaptors suggest that microneme targeting may exploit the same precursor compartment involved in rhoptry targeting. Nuclear-encoded proteins destined for the apicoplast exhibit a bipartite NH₂-terminal domain (Roos et al., 1999; DeRocher et al., 2000; Waller et al., 2000; Yung et al., 2001), mediating transport first into the secretory pathway using a classical secretory signal sequence, and subsequently into the apicoplast using a plastid–transit peptide akin to that found in plants. Whether all secreted proteins transit this organelle after exit from the Golgi remains to be determined, as does the ultimate destination of products produced in the apicoplast (dashed black arrows). A, apicoplast; DG, dense granule; E, endosome; Mn, micronemes; PC, precursor compartment; Rh, rhoptries.