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Proteasome activity in a naïve mosquito cell line infected with Wolbachia pipientis wAlbB

Author:
Fallon, Ann M., Witthuhn, Bruce A.
Source:
In vitro cellular & developmental biology 2009 v.45 no.8 pp. 460-466
ISSN:
1071-2690
Subject:
Aedes albopictus, Western blotting, Wolbachia pipientis, biochemical pathways, cultured cells, cysteine, gels, inoculum, metabolism, methionine, monoclonal antibodies, proteasome endopeptidase complex, sodium dodecyl sulfate, tandem mass spectrometry, ubiquitin
Abstract:
We used Wolbachia pipientis strain wAlbB from Aedes albopictus Aa23 cells to infect clonal Ae. albopictus TK-6 cells, which are resistant to 5-bromodeoxyuridine. Infected TK-6 cells were cultured in medium containing 5-bromodeoxyuridine to select against Aa23 cells that might have persisted in the inoculum. Infected TK-6 lines retained the Wolbachia infection for 5 mo, indicating that their metabolic processes support Wolbachia growth and multiplication. To investigate early events after Wolbachia infection, we labeled infected cells with ³⁵S[methionine/cysteine]. Patterns of labeled proteins on sodium dodecyl sulfate gels were similar in control and infected cells, with the exception of a 29-kDa protein. Tandem mass spectrometry revealed that the 29-kDa band included α and β subunits of the 26S proteasome. Independent confirmation of the up-regulation of the proteasome was established by probing Western blots with a monoclonal antibody to the proteasome-associated co-factor, ubiquitin. Wolbachia's loss of metabolic pathways for the synthesis of most amino acids and retention of pathways for their uptake and metabolism suggest that proteasome activation provides a mechanism whereby controlled degradation of intracellular host proteins would increase availability of amino acids to support establishment and maintenance of the Wolbachia infection.
Agid:
2198543