Quantitative Profiling for Substrates of the Mitochondrial Presequence Processing Protease Reveals a Set of Nonsubstrate Proteins Increased upon Proteotoxic Stress

The
majority of mitochondrial preproteins are targeted via N-terminal
presequences that are cleaved upon import into the organelle. The
essential mitochondrial processing protease (MPP) is assumed to cleave
the majority of incoming precursors; however, only a small fraction
of mitochondrial precursors have been experimentally analyzed limiting
the information on MPP recognition and substrate specificity. Here
we present the first systematic approach for identification of authentic
MPP substrate proteins using a temperature-sensitive mutant of the
MPP subunit Mas1. Inactivation of MPP at nonpermissive temperature
leads to accumulation of immature precursors in mitochondria, which
were measured by quantitative N-terminal ChaFRADIC. This led to the
identification of 66 novel MPP substrates. Deduction of the cleaved
presequences determines arginine in position −2 of the cleavage
site as a main factor for MPP recognition. Interestingly, a set of
nonprocessed proteins was also increased in mas1 mutant
mitochondria. Additionally, mas1 mitochondria respond
to temperature elevation with an increase in membrane potential and
oxygen consumption. These changes might indicate that mas1 cells exert a response to balance the proteotoxic stress induced
by MPP dysfunction.