Abstract
The polymorphic mitochondrial translation product varl has been analyzed by one- and two-dimensional gel electrophoresis and by proteolytic cleavage of the radiolabeled product. Different apparent molecular weight forms of varl ranging between 40,000 and 44,000 show “normal” migration behavior as a function of the acrylamide concentration on sodium dodecyl sulfate- polyacrylamide gels. Comparisons of peptide fragment patterns generated by digestion of different molecular weight forms of varl with papain and a protease from Staphylococcus aureus V8 show considerable fragment homology; some partial fragments retain the molecular weight differences of the undigested product; others shown no homology and suggest the presence of unique cleavage sites. Analysis on a two-dimensional system consisting of electrophoresis in the first dimension on acid-urea gels (Mets, L., and Bogorad, L., (1974) Anal. Biochem. 57, 200-220) and in the second dimension on sodium dodecyl sulfate-polyacrylamide gels, show that varl behaves as a basic protein migrating between the cytoplasmic large subunit ribosomal proteins L2 and L3. Varl is the only major labeled polypeptide species to be resolved in this two dimensional gel system when cells are labeled with 3sS042- in uiuo in the presence of cycloheximide. By analyzing yeast strains containing different molecular weight forms of varl, we show that the protein is specifically associated with the 38 S mi- tochondrial ribosomal subunit. By Coomassie blue staining, it appears to be present in amounts roughly equivalent to the other proteins of the 38 S subunit and may be an integral ribosomal protein since it is not removed by a high salt wash. When cells are labeled in viva with 35S042- in the absence of inhibitors, most, if not all, of the varl is associated with the 38 S subunit. Between 20% and 80% of the varl can be found in the postmitochondrial supernatant fraction. Marker en- zyme distribution studies suggest that this “extra mi- tochondrial” varl is released by mechanical damage of the mitochondria.
Highlights
From the Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235
As we have previously noted [6, 7], wild type yeast strains which differ in the molecular weight form of varl show no obvious phenotypic alterations which can be attributed in any way to a particular varl species
The possibility remained, that the form of varl which we identify in any particular yeast strain by the procedure of selective labeling of mitochondrial translation products in Go, is only some intermediate form or side product, and that the putative mature and functioning varl is processed in all strains to exactly the same size
Summary
Yeast Strains-Isonuclear diploids with different forms of varl were obtained from a cross between strains 5DSS After two washes with HzO, the cell pellets were resuspended in AMT-500 at about 1 g wet weight per ml of buffer and broken by shaking for 3 min with glass beads (0.45 to 0.5 mm) in a Bronwill homogenizer. Electrophoresis of mitochondrial translation products in SDS-polyacrylamide gels containing a horizontal gradient of acrylamide was carried out according to the procedure of Beckendorf and Kafatos [17]. Cells from a lo-ml culture in mid-log phase were labeled with ““SOI”- in the presence of cycloheximide as described [6, 16], washed with H20, and broken with glass beads in the Bronwill homogenizer in 200 al of buffer containing 50 mM NCLCl, 10 mM MgC12, 10 mru. ProteinDeterminations-Thesweere carried out by a modification of the procedure of Lowry etal. [25] using deoxycholate as carrier for trichloroacetic acid precipitation and washing of protein pellets
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