Lysine residues at positions 234 and 236 in yeast porin are involved in its assembly into the mitochondrial outer membrane

M. D. Smith, M. Petrak, P. D. Boucher, K. N. Barton, L. Carter, G. Reddy, E. Blachly- Dyson, M. Forte, J. Price, K. Verner, R. B. McCauley

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Abstract

Various point mutations of lysyl residues in yeast mitochondrial porin (283 residues) were tested for their ability to assemble in vitro into the outer membranes of intact yeast mitochondria. Assembly was evaluated by protection from proteinases. The extent of assembly of two of the mutants, K234E and K236E porins, was much less than for wild-type in either post- translational or co-translational assembly assays. Lysine to glutamate mutants at other positions and K234R porin assembled as well as wild-type, but K234Q porin was poorly inserted. When both Lys-234 and Lys-236 were mutated, K234R/K236R porin was inserted better than K234Q/K236Q porin, which was inserted better than K234E/K236E; however, none of these mutants assembled as well as wild-type porin. It was concluded that optimal assembly of yeast porin depended on the presence of positively charged residues at both positions 234 and 236 and a lysine at one of these positions. After undergoing the assembly reaction, mutants that were vulnerable to proteinase K (i.e. K234E, K234Q, and K236E porins) seemed to be incompletely digested and were, to varying degrees, resistant to extraction by Na2CO3 (pH 11.5). These experiments suggested that these mutants were incompletely inserted into the outer membrane. Both Lys-234 and Lys-236 are included in an internal pentapeptide, VKAKV, that is conserved in porins from protists, plants, and animals, and it is possible that, at least, the lysines in this tract are one of the signals for the membrane assembly of these proteins.

Original languageEnglish (US)
Pages (from-to)28331-28336
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number47
DOIs
StatePublished - Jan 1 1995

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Smith, M. D., Petrak, M., Boucher, P. D., Barton, K. N., Carter, L., Reddy, G., Blachly- Dyson, E., Forte, M., Price, J., Verner, K., & McCauley, R. B. (1995). Lysine residues at positions 234 and 236 in yeast porin are involved in its assembly into the mitochondrial outer membrane. Journal of Biological Chemistry, 270(47), 28331-28336. https://doi.org/10.1074/jbc.270.47.28331