TY - JOUR
T1 - Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3
AU - Wallbank, Bethan A.
AU - Dominicus, Caia S.
AU - Broncel, Malgorzata
AU - Legrave, Nathalie
AU - Kelly, Gavin
AU - MacRae, James I.
AU - Staines, Henry M.
AU - Treeck, Moritz
N1 - Publisher Copyright:
© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.
PY - 2019/5
Y1 - 2019/5
N2 - Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.
AB - Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.
UR - http://www.scopus.com/inward/record.url?scp=85057223906&partnerID=8YFLogxK
U2 - 10.1111/mmi.14156
DO - 10.1111/mmi.14156
M3 - Article
C2 - 30402958
AN - SCOPUS:85057223906
SN - 0950-382X
VL - 111
SP - 1167
EP - 1181
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -