5 research outputs found
Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery
Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Δ mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane
Comparison of Alternative MS/MS and Bioinformatics Approaches for Confident Phosphorylation Site Localization
Over the past years, phosphoproteomics
has advanced to a prime
tool in signaling research. Since then, an enormous amount of information
about <i>in vivo</i> protein phosphorylation events has
been collected providing a treasure trove for gaining a better understanding
of the molecular processes involved in cell signaling. Yet, we still
face the problem of how to achieve correct modification site localization.
Here we use alternative fragmentation and different bioinformatics
approaches for the identification and confident localization of phosphorylation
sites. Phosphopeptide-enriched fractions were analyzed by multistage
activation, collision-induced dissociation and electron transfer dissociation
(ETD), yielding complementary phosphopeptide identifications. We further
found that MASCOT, OMSSA and Andromeda each identified a distinct
set of phosphopeptides allowing the number of site assignments to
be increased. The postsearch engine SLoMo provided confident phosphorylation
site localization, whereas different versions of PTM-Score integrated
in MaxQuant differed in performance. Based on high-resolution ETD
and higher collisional dissociation (HCD) data sets from a large synthetic
peptide and phosphopeptide reference library reported by Marx et al.
[<i>Nat. Biotechnol</i>. <b>2013</b>, <i>31</i> (6), 557–564], we show that an Andromeda/PTM-Score probability
of 1 is required to provide an false localization rate (FLR) of 1%
for HCD data, while 0.55 is sufficient for high-resolution ETD spectra.
Additional analyses of HCD data demonstrated that for phosphotyrosine
peptides and phosphopeptides containing two potential phosphorylation
sites, PTM-Score probability cutoff values of <1 can be applied
to ensure an FLR of 1%. Proper adjustment of localization probability
cutoffs allowed us to significantly increase the number of confident
sites with an FLR of <1%.Our findings underscore the need for the
systematic assessment of FLRs for different score values to report
confident modification site localization