Helmut E. Meyer

Sam35 of the Mitochondrial Protein Sorting and Assembly Machinery Is a Peripheral Outer Membrane Protein Essential for Cell Viability

The mitochondrial outer membrane contains two integral proteins essential for cell viability, Tom40 of the translocase of the outer membrane (TOM complex) and Sam50 of the sorting and assembly machinery (SAM complex). Here we report the identification of Sam35, the first peripheral mitochondrial outer membrane protein that is essential for cell viability. Sam35 (encoded by the Saccharomyces cerevisiae ORF YHR083w) is a novel subunit of the SAM complex and is crucial for the assembly pathway of outer membrane β-barrel proteins, such as the precursors of Tom40 and porin. Sam35 is not required for the import of inner membrane or matrix targeted proteins. The presence of two essential proteins in the SAM complex, Sam35 and Sam50, indicates that it plays a central role in mitochondrial biogenesis.

Next-Generation Trapping of Protease Substrates by Label-Free Proteomics

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.