Towards unveiling the Photoactive Yellow Proteins: characterization of a halophilic member and a proteomic approach to study light responses

The family of blue-light absorbing Photoactive Yellow Proteins (PYPs) has become an attractive model system to study the molecular events related to light perception. PYP covalently binds a p-hydroxycinnamic acid chromophore, which is fine-tuned for efficient light absorption and initiates a photocycle by a trans to cis isomerization. PYPs were originally discovered in a number of halophilic proteobacteria, but the pyp gene distribution actually spans a wider spectrum of both phototrophic as well as non-photosynthetic bacteria. PYP from Salinibacter ruber is shown to be the first example of a truly halophilic PYP that is stabilized by ionic strength. Furthermore, illumination with blue light ignites a cyclic series of dark reactions including the typical intermediates I1, I2 and I2’, which suggests that it is functional although the recovery to the dark-adapted state was observed to be the slowest of any PYP. More interestingly, PYP from Salinibacter ruber contains an unusual 31-residue N-terminal extension which appears to be disordered relative to the remainder of the protein and which induces a unique dimerization of the photosensor with no precedents described before with any of the other published PYPs. Truncation of the N-terminal extension does not seem to influence the photochemical properties, and no structural motif or functional relevance towards signaling could thus far be established. PYP can also be part of multi-domain proteins such as Ppr from the phototroph Rhodospirillum centenum, where it forms the N-terminal domain that is followed by a central bacteriophytochrome (Bph) domain and by a C-terminal histidine kinase domain. The PYP domain acts as a blue-light switch reversing the effects of red light on the Bph domain, resulting in a light-regulated histidine kinase activity of Ppr. Comparative proteomics of wild-type Rh. centenum relative to a ppr gene deletion mutant revealed complex alterations in response to actinic blue- (390 - 510 nm) and red- (>600 nm) light conditions during photosynthetic growth. Differentially regulated proteins provide indications that the Ppr-mediated photoresponse involves an increased acetyl-CoA pool causing a shift in the lipid metabolism in favor of polyketide biosynthesis. However, the nature of this adaptation remains unclear and requires further study

Differential protein expression in the honey bee head after a bacterial challenge

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A Reciprocal N-15-labeling proteomic analysis of expanding arabidopsis leaves subjected to osmotic stress indicates importance of mitochondria in preserving plastid functions

Plants respond to environmental stress by dynamically reprogramming their growth. Whereas stress onset is accompanied by rapid growth inhibition leading to smaller organs, growth will recover and adapt once the stress conditions become stable and do no threaten plant survival. Here, adaptation of growing Arabidopsis thaliana leaves to mild and prolonged osmotic stress was investigated by means of a complete metabolic labeling strategy with the N-15-stable isotope as a complement to a previously published transcript and metabolite profiling. Global analysis of protein changes revealed that plastidial ATPase, Calvin cycle, and photorespiration were down-regulated, but mitochondrial ATP synthesis was up-regulated, indicating the importance of mitochondria in preserving plastid functions during water stress. Although transcript and protein data correlated well with the stable and prolonged character of the applied stress, numerous proteins were clearly regulated at the post-transcriptional level that could, at least partly, be related to changes in protein synthesis and degradation. In conclusion, proteomics using the N-15 labeling helped understand the mechanisms underlying growth adaptation to osmotic stress and allowed the identification of candidate genes to improve plant growth under limited water

The growing family of photoactive yellow proteins and their presumed functional roles

For several years following the discovery and characterization of the first PYP, from Halorhodospira halophila, it was thought that this photoactive protein was quite unique, notwithstanding the isolation of two additional examples in rapid succession. Mainly because of genomic and metagenomic analyses, we have now learned that more than 140 PYP genes occur in a wide variety of bacteria and metabolic niches although the protein has not been isolated in most cases. The amino acid sequences and physical properties permit their organization into at least seven groups that are also likely to be functionally distinct. Based upon action spectra and the wavelength of maximum absorbance, it was speculated nearly 20 years ago but never proven that Hr. halophila PYP was involved in phototaxis. Nevertheless, in only one instance has the functional role and interaction partner for a PYP been experimentally proven, in Rs. centenum Ppr. Genetic context is one of several types of evidence indicating that PYP is potentially involved in a number of diverse functional roles. The interaction with other sensors to modulate their activity stands out as the single most prominent role for PYP. In this review, we have attempted to summarize the evidence for the functional roles and interaction partners for some 26 of the 35 named species of PYP, which should be considered the basis for further focused molecular and biochemical research

In Chlamydomonas, the Loss of Nd5 Subunit Prevents the Assembly of Whole Mitochondrial Complex I and Leads to the Formation of a Low Abundant 700 Kda Subcomplex

In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the beta membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I-complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex