Current Genetics

, Volume 20, Issue 1, pp 87–90

Over-expression, purification and determination of the proteolytic processing site of the yeast mitochondrial CBS1 protein

  • A. Körte
  • U. Michaelis
  • F. Lottspeich
  • G. Rödel
Original Articles

DOI: 10.1007/BF00312770

Cite this article as:
Körte, A., Michaelis, U., Lottspeich, F. et al. Curr Genet (1991) 20: 87. doi:10.1007/BF00312770

Summary

Yeast transformats harboring the CBS1 gene under the control of the strong ADC1 promoter on a high copy number plasmid express the mitochondrial CBS1 protein at artificially high levels. Over-expressed protein is imported into mitochondria and correctly processed to vield the mature mitochondrial 23.5 kDa form, but differs in its solubility properties from CBS1 in wild-type mitochondria. It forms insoluble protein aggregates, which are refractory to solubilization with 1% Taurodeoxycholate. We exploited this observation to separate CBS1 from the bulk of mitochondrial proteins and to isolate CBS1 after SDS gel electrophoresis. Determination of the amino-terminal amino acids of the purified protein reveals that the mature CBS1 protein starts with Ile30, at the characteristic distance of +2 amino acids from an arginine residue (Arg28). The cleavage site shows a remarkable homology to that of subunit 9 of the F0F1 ATPase from Neurospora crassa.

Key words

CBS1 proteinProtein purificationMitochondrial presequenceProtein sequencing

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • A. Körte
    • 1
  • U. Michaelis
    • 1
  • F. Lottspeich
    • 2
  • G. Rödel
    • 3
  1. 1.Lehrstuhl für GenetikUniversität MünchenMünchenFederal Republic of Germany
  2. 2.Max-Planck-Institut für BiochemicGenzentrumMartinsriedFederal Republic of Germany
  3. 3.Labor für Molekulare Biologie und Allgemeine Pathologie, Institut für PathologieUniversität UlmMünchen 45Federal Republic of Germany