Mutational analysis of a signal sequence required for protein secretion in Leishmania major.
In eukaryotes, amino-terminal extensions, signal sequences, mediate the translocation of lysosomal, membrane and secreted proteins into the lumen of the endoplasmic reticulum (ER). Structure/function studies indicate that eukaryotic signal sequences are composed of 3 distinct domains, a positively charged amino-terminal domain, a central hydrophobic domain, and a polar carboxy-terminal domain. In an attempt to better understand protein trafficking in Leishmania we have constructed strains of Leishmania major that secrete an exogenous protein, interferon-gamma (IFN-gamma), under the control of a mammalian signal sequence. In this report we present a mutational analysis of this signal sequence. Deletion of the entire signal sequence or the hydrophobic core region prevents secretion of IFN-gamma and results in cytoplasmic expression of the protein. Mutations in the amino-terminal domain indicate that a net positive charge is not required for efficient secretion of IFN-gamma. Mutations in the carboxy-terminal domain are more complex and display two phenotypes, either they prevent expression of IFN-gamma or they have no effect on protein secretion. These results indicate that the function of the signal sequence in targeting proteins to the ER in Leishmania is similar to that observed in yeasts and higher eukaryotes and suggests that the Leishmania protein secretory apparatus may also be similar.
Tobin
J F
JF
Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02215.
Wirth
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan; D007371:Interferon-gamma / Q000378:metabolism*; D018320:Leishmania major / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D021382:Protein Sorting Signals; D007371:Interferon-gamma