Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin.
The LmjF01.0030 gene of Leishmania major Friedlin, annotated as 'MCAK-like', was confirmed as a kinesin with an internally located motor domain and termed LmjKIN13-1. Both the native form of the protein and a green fluorescent protein (GFP)-fused recombinant version were shown to be exclusively intranuclear, and, more specifically, to localize to the spindle and spindle poles. Cell cycle-dependent regulation of the protein levels was demonstrated using synchronized Leishmania cells: LmjKIN13-1 was highly abundant in the G2+M phase and present at very low levels after mitosis. Altogether, these features suggest that this protein participates in mitosis. The construction of systematic deletion mutants allowed the localization of the primary sequence regions responsible for nuclear targeting on the one hand, and for cell cycle-dependent variations on the other hand. A 42-amino-acid region of the carboxy(C)-terminal domain mediates nuclear import and could be defined as an atypical nuclear localization signal. Protein level regulation during the cell cycle was shown to also depend upon the C-terminal domain, where apparently redundant degradation signals are present. Putative degradation signals appear to be present on both sides and inside the nuclear localization signal. Further experiments strongly suggest a role for the ubiquitin/proteasome pathway in this cell cycle-dependent regulation. These data underline the importance of post-translational regulation of protein abundance in this ancestral eukaryote where transcriptional regulation seems to be rare or near absent.
Dubessay
Pascal
P
UMR5093 CNRS/Université Montpellier I Génome et Biologie Moléculaire des Protozoaires Parasites, Laboratoire de Parasitologie-Mycologie, Faculté de Médecine, 163 Rue Auguste Broussonet, 34090 Montpellier, France.
Blaineau
Christine
C
Bastien
Patrick
P
Tasse
Lena
L
Van Dijk
Juliette
J
Crobu
Lucien
L
Pagès
Michel
M
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D016547:Kinesins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018320:Leishmania major / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D019913:Nuclear Localization Signals; D061988:Proteasome Inhibitors; D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex; D016547:Kinesins