PSSA-2, a membrane-spanning phosphoprotein of Trypanosoma brucei, is required for efficient maturation of infection.

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T(305)), a modification that depends on the presence of TbMAPK4. Mutation of T(305) to alanine (T(305)A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T(305)D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein's structure and the effects of mutation of T(305) on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite's decision to divide, differentiate or migrate.

The procyclin-associated genes of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

EP and GPEET procyclins are the major surface glycoproteins of Trypanosoma brucei in the midgut of tsetse flies (Glossina spp.). The procyclin genes are located at the beginning of polycistronic transcription units and are followed by at least one procyclin-associated gene (PAG). The EP/PAG1 locus on one copy of chromosome X begins with the three genes EP1, EP2 and PAG1; the end of this unit has not been characterized previously. The EP/PAG2 locus on the other copy of chromosome X contains the same procyclin genes followed by PAG2 and PAG4. Here we show that the EP/PAG1 locus in AnTat1.1 has to be extended by three more PAGs, which we named PAG5, PAG2* and PAG4. The EP/PAG2 locus most likely evolved from the EP/PAG1 locus by deletion of a fragment from within PAG1 to PAG2*. The procyclin loci on the two copies of chromosome VI are indistinguishable, and contain the genes GPEET, EP3, PAG3 and GRESAG2.1. The mRNA levels of PAG1, PAG2 and PAG3 are transiently increased during differentiation of bloodstream forms to procyclic forms. Unexpectedly, procyclic forms of a PAG knockout clone lacking all eight PAGs in the procyclin loci were transmissible by Glossina morsitans. Furthermore, the deletion mutant could still establish midgut infections when competing with a tagged clone with the full complement of PAGs. Cyclical transmission was also possible when tsetse flies were infected with bloodstream forms of the deletion mutant, demonstrating that the PAGs are not essential for the differentiation of bloodstream to procyclic forms in vivo.

Mitotic and stress-induced phosphorylation of HsPI3K-C2alpha targets the protein for degradation.

Activation of the phosphoinositide 3-kinases (PI 3-kinases) has been implicated in multiple cellular responses such as proliferation and survival, membrane and cytoskeletal reorganization, and intracellular vesicular trafficking. The activities and subcellular localization of PI 3-kinases were shown to be regulated by phosphorylation. Previously we demonstrated that class II HsPIK3-C2alpha becomes phosphorylated upon inhibition of RNA pol II-dependent transcription (Didichenko, S. A., and Thelen, M. (2001) J. Biol. Chem. 276, 48135-48142). In this study we investigated cell cycle-dependent and genotoxic stress-induced phosphorylation of HsPIK3-C2alpha. We find that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of the cell cycle. Stress-dependent and mitotic phosphorylation of HsPIK3-C2alpha occurs on the same serine residue (Ser259) within a recognition motif for proline-directed kinases. Mitotic phosphorylation of HsPIK3-C2alpha can be attributed to Cdc2 activity, and stress-induced phosphorylation of HsPIK3-C2alpha is mediated by JNK/SAPK. The protein level of HsPIK3-C2alpha is regulated by proteolysis in a cell cycle-dependent manner and in response of cells to stress. Phosphorylation appears to be a prerequisite for proteasome-dependent degradation of HsPIK3-C2alpha and may therefore contribute indirectly to the regulation of the activity of the kinase.