Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.

Abstract

Recent results with N. crassa show that 1 protein (S-5, MW 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria. Neurospora mit ribosomal proteins were analyzed by 2-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 M KCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI [isoelectric point] > 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol rapidly inhibited the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits. Two-dimensional gel analysis shows that the more slowly sedimenting CAP-30S particles are deficient in S-5 and in several other proteins; these proteins are present in normal concentrations in mature small subunits from the same cells. S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol; S-5 may play a role in the assembly of mit small subunits. Apparently S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and 6 additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. [Poky] Mit small subunits are deficient in S-5 and contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].