Disruption of the zinc finger motifs in the Leishmania tarentolae LC-4 (=TbMP63) L-complex editing protein affects the stability of the L-complex.
The uridine insertion/deletion editing complex, which we have termed the L-complex, is composed of at least 16 polypeptides stabilized entirely by protein-protein interactions. Three L-complex proteins contain zinc finger motifs that could be involved in these interactions. In Leishmania these proteins are labeled LC-1, LC-4, and LC-7b, and the orthologs in Trypanosoma brucei are labeled MP81, MP63, and MP42. Overexpression of TAP-tagged LC-4 in Leishmania tarentolae led to a partial localization of the protein in the L-complex together with the endogenous LC-4 protein, suggesting at least a dimeric organization. Disruption of zinc fingers 1 or 2 (ZnF-1 and ZnF-2) in the tagged LC-4 protein was performed by mutation of the two zinc-binding cysteines to glycines. Disruption of ZnF-1 led to a partial growth defect and a substantive breakdown of the L-complex, whereas disruption of ZnF-2 had no effect on cell growth and caused a partial breakdown of the L-complex. A close interaction of LC-4 with 2-4 proteins, including REL1 (RNA ligase) and LC-3, was suggested by chemical crosslinking and co-immunoprecipitation experiments. Our results suggest that both ZnF-1 and ZnF-2 in LC-4 play a role in protein-protein interactions and indicate that the LC-4 subcomplex may be required for formation or stability of the entire L-complex.
Kang
Xuedong
X
Howard Hughes Medical Institute, Los Angeles, CA 90095, USA.
Falick
Arnold M
AM
Nelson
Robert E
RE
Gao
Guanghan
G
Rogers
Kestrel
K
Aphasizhev
Ruslan
R
Simpson
Larry
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D017125:Genes, Protozoan; D007891:Leishmania / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D016054:DNA, Protozoan; D046911:Macromolecular Substances; D015800:Protozoan Proteins; D011994:Recombinant Proteins