Gene cloning and cellular localization of a membrane-bound acid phosphatase of Leishmania mexicana.
In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70000-72000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane alpha-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.
Wiese
M
M
Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Tübingen, Germany. wiese@gen.mpib-tuebingen.mpg.de
Berger
O
O
Stierhof
Y D
YD
Wolfram
M
M
Fuchs
M
M
Overath
P
P
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D011992:Endosomes / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D017125:Genes, Protozoan; D007894:Leishmania mexicana / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D015388:Organelles / Q000201:enzymology*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D000135:Acid Phosphatase