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Cell cycle‐dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin‐13 kinesin
Author(s) -
Dubessay Pascal,
Blaineau Christine,
Bastien Patrick,
Tasse Lena,
Van Dijk Juliette,
Crobu Lucien,
Pagès Michel
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.05013.x
Subject(s) - biology , nuclear localization sequence , mitosis , cell cycle , microbiology and biotechnology , nuclear transport , nuclear export signal , proteasome , nuclear protein , ubiquitin , protein degradation , kinesin , cell nucleus , genetics , microtubule , gene , cytoplasm , transcription factor
Summary The Lmj F01.0030 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: Lmj KIN13‐1 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.