title: Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids.
abstract: Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3 and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.
authors: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. rajgaji@umich.edu
authors: Rajshekhar Y
authors: RY
authors: Gaji
authors: Halley P
authors: HP
authors: Flammer
authors: Vern B
authors: VB
authors: Carruthers
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D006801:Humans; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014122:Toxoplasma / Q000166:cytology / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D000596:Amino Acids; D011498:Protein Precursors; D015800:Protozoan Proteins; C450759:TgM2AP protein, Toxoplasma gondii