A Toxoplasma Palmitoyl Acyl Transferase and the Palmitoylated Armadillo Repeat Protein TgARO Govern Apical Rhoptry Tethering and Reveal a Critical Role for the Rhoptries in Host Cell Invasion but Not Egress
2013; 9 (2)
Toxoplasma ISP4 is a central IMC Sub-compartment Protein whose localization depends on palmitoylation but not myristoylation
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
2012; 184 (2): 99-108
Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.
View details for DOI 10.1371/journal.ppat.1003162
View details for Web of Science ID 000315648900016
View details for PubMedID 23408890
View details for PubMedCentralID PMC3567180
Apicomplexan parasites utilize a peripheral membrane system called the inner membrane complex (IMC) to facilitate host cell invasion and parasite replication. We recently identified a novel family of Toxoplasma IMC Sub-compartment Proteins (ISP1/2/3) that localize to sub-domains of the IMC using a targeting mechanism that is dependent on coordinated myristoylation and palmitoylation of a series of residues in the N-terminus of the protein. While the precise functions of the ISPs are unknown, deletion of ISP2 results in replication defects, suggesting that this family of proteins plays a role in daughter cell formation. Here we have characterized a fourth ISP family member (ISP4) and discovered that this protein localizes to the central IMC sub-compartment, similar to ISP2. Like ISP1/3, ISP4 is dispensable for the tachyzoite lytic cycle as the disruption of ISP4 does not produce any gross replication or growth defects. Surprisingly, targeting of ISP4 to the IMC membranes is dependent on residues predicted for palmitoylation but not myristoylation, setting its trafficking apart from the other ISP proteins and demonstrating distinct mechanisms of protein localization to the IMC membranes, even within a family of highly related proteins.
View details for DOI 10.1016/j.molbiopara.2012.05.002
View details for Web of Science ID 000306626300005
View details for PubMedID 22659420
View details for PubMedCentralID PMC3383393