Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit.
Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.
*Instituto de Parasitología y Biomedicina López Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n 18016-Armilla, Granada, Spain.
Sebastián
S
García-Sánchez
*Instituto de Parasitología y Biomedicina López Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n 18016-Armilla, Granada, Spain.
María P
MP
Sánchez-Cañete
*Instituto de Parasitología y Biomedicina López Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n 18016-Armilla, Granada, Spain.
Francisco
F
Gamarro
*Instituto de Parasitología y Biomedicina López Neyra, IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n 18016-Armilla, Granada, Spain.
Santiago
S
Castanys
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D005110:Extracellular Space / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D018314:Leishmania infantum; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000502:physiology*; D011817:Rabbits; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
C427246:CDC50 protein, S cerevisiae; D029721:Drosophila Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C480230:miltefosine transporter, Leishmania donovani