8 research outputs found
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 ā2 of the cleavage
site as a main factor for MPP recognition. Interestingly, a set of
nonprocessed proteins was also increased in <i>mas1</i> mutant
mitochondria. Additionally, <i>mas1</i> mitochondria respond
to temperature elevation with an increase in membrane potential and
oxygen consumption. These changes might indicate that <i>mas1</i> cells exert a response to balance the proteotoxic stress induced
by MPP dysfunction
Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive NāTerminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55
We present a novel straightforward method for enrichment of N-terminal
peptides, utilizing charge-based fractional diagonal chromatography
(ChaFRADIC). Our method is robust, easy to operate, fast, specific,
and more sensitive than existing methods, enabling the differential
quantitation of 1459 nonredundant N-terminal peptides between two <i>S. cerevisiae</i> samples within 10 h of LCāMS, starting
from only 50 Ī¼g of protein per condition and analyzing only
40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial
proteins from wild-type and icp55Ī yeast (30 Ī¼g each).
Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent
cleavage of signal sequences, removes a single amino acid from a specific
set of proteins according to the N-end rule. Using ChaFRADIC we identified
36 icp55 substrates, 14 of which were previously unknown, expanding
the set of known icp55 substrates to a total of 52 proteins. Interestingly,
a novel substrate, Isa2, is likely processed by Icp55 in two consecutive
steps and thus might represent the first example of a triple processing
event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful
and practicable tool for protease and peptidase research, providing
the sensitivity to characterize even samples that can be obtained
only in small quantities