Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Oct 1;29(20):2386–2396. doi: 10.1091/mbc.E18-04-0227

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2018 Möller-Hergt et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

PMC Copyright notice

FIGURE 2: — Mrx15 is an inner membrane mitochondrial protein that interacts with the large ribosomal subunit. (A) Western blot of mitochondrial extracts containing a chromosomally PA-tagged variant of Mrx15. Mitochondria were lysed in 10 mM EDTA for Mg depletion and separated afterward on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrps5), and PA for Mrx15 detection. (B) Abundancy of PA-tagged variants of the SSU (Mrp1PA), LSU (Mhr1PA), and Mrx15 were compared by Western blotting. Aconitase (Aco1) and Mrpl4 were used as loading controls. (C) Carbonate extraction of mitochondrial extracts. After incubation with either 0.2 M Na₂CO₃ or 0.2 M NaCl, the membrane and soluble fractions of mitochondrial extracts were separated by high-velocity centrifugation. Supernatant and pellet fractions were probed for membrane (Cbp4) and soluble (Aco1) proteins and PA for Mrx15 detection. (D) Protease protection assay of mitochondria (Mitos), mitoplasts, and lysed mitochondria. Mitochondria, mitoplasts created by hypotonic swelling of mitochondria, and detergent-lysed mitochondria were treated for 20 min with proteinase K (PK). Afterward, a Western blot of each sample was probed for proteins of the outer membrane (Tom70), inner membrane (Cbp4), and the matrix (Mrpl36). The PA antibody was used for detection of the PA-tagged variant of Mrx15. (E) Schematic representation of full-length Mrx15 tagged with PA and a C-terminal truncation variant. Cell extracts of strains grown to log phase containing either variant were probed for PA to determine abundance of each construct. (F) Western blot of mitochondrial extracts containing C-terminally truncated variant of Mrx15. Interaction with the mitoribosome was tested by Mg depletion and separation of ribosomal subunits on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrp1), and PA for Mrx15 detection. (G) Mrx15 inner membrane topology. According to our results, the Mrx15 protein is a component of the inner mitochondrial membrane. The N- and C-termini of the protein face the matrix. The C-terminal domain is necessary for interaction with the large ribosomal subunit. IMS, intermembrane space; Mitopl., mitoplasts; P, pellet; SN, supernatant; T, total; TM, transmembrane segment; WT, wild type.