Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag

Vasant Muralidharan, Anna Oksman, Mari Iwamoto, Thomas J. Wandless, Daniel E. Goldberg

Research output: Contribution to journalArticle

78 Scopus citations

Abstract

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.

Original languageEnglish
Pages (from-to)4411-4416
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number11
DOIs
StatePublished - Mar 15 2011

Keywords

  • Amino acid repeat
  • Conditional expression
  • Malaria
  • Polyasparagine
  • Trinucleotide repeat

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