The proteolytic processing of organellar proteins

Biogenesis of the chloroplast involves the activities of both the nuclear and chloroplast genomes. Nuclear-encoded chloroplast proteins are synthesised on free ribosomes in the cytosol and imported into the chloroplast post-translationally. Protein uptake is directed by an N-terminal presequence which is removed after import by specific processing peptidases. Thylakoid lumen proteins must cross three membranes to reach their site of function and have a composite presequence to enable their correct localisation, consisting of an envelope transit domain (ETD) and a thylakoid transfer domain (TTD). The ETD is removed after translocation across the envelope by a stromal processing peptidase (SPP) to produce an intermediate-sized protein and the TTD then allows transport into the thylakoid lumen where it is removed by a thylakoidal processing peptidase (TPP). SPP is a soluble, stromal- located metallopeptidase which also removes the presequences from stromal proteins. Despite its apparently high specificity, there is very little sequence similarity around SPP cleavage sites in stromal and thylakoidal proteins, and little is known about the structural features which SPP recognises. SPP was partially purified from pea chloroplasts and the SPP cleavage site within the presequences of three thylakoid lumen proteins was determined by radiosequencing of the cleavage products with the two-fold aim of identifying the residues around the cleavage site which SPP may recognise and of delineating the ETD and TTD regions of the presequences, allowing their comparison with signal sequences. This information was then used to create four mutant thylakoid lumen precursor proteins by site-directed mutagenesis with altered SPP processing sites. The four mutant proteins all showed a reduced rate and efficiency of processing in an organelle-free time course assay, although all were imported into chloroplasts, correctly localised and processed to the mature size in an in vitro assay. Although SPP has usually been considered to be highly specific for impoited chloroplast precursor proteins, it has recently been suggested that it may also process mitochondrial precursor proteins, which are cleaved in vivo and in vitro to the mature size by the mitochondrial processing peptidase (MPP). These two peptidases were therefore compared in terms of substrate specificity and mechanism by assaying the activity of the two enzymes against chloroplast and mitochondrial precursor proteins. MPP did not process any of the chloroplast precursor proteins available, suggesting that this enzyme is indeed highly specific for mitochondrial precursor proteins. A partially-purified SPP preparation cleaved the mitochondrial precursor proteins tested to a smaller size; however, the site of cleavage in at least some of these proteins was different to the authentic MPP cleavage site, with the stromal preparation cleaving N- terminal to MPP. This stromal processing activity was not due to mitochondrial contamination and evidence from inhibitor sensitivities and column chromatography suggests that SPP cleaves both chloroplast and mitochondrial precursor proteins. Hie SPP processing site within the presequences of two mitochondrial proteins was determined by radiosequencing and compared with the chloroplast cleavage sites, and this data should enable further analysis to determine the features which constitute a site which can be recognised by SPP. An SPP activity was also partially-purified from Chlamydomonas reinhardtii and shown to be located in the stroma. This activity is able to process chloroplast precursor proteins from C. reinhardtii and pea to an intermediate or mature size, emphasising the similarity of the specificity of SPP from these two species

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

Hydrogen sulfide: From a toxic molecule to a key molecule of cell life

Hydrogen sulfide (H2S) has always been considered toxic, but a huge number of articles published more recently showed the beneficial biochemical properties of its endogenous production throughout all regna. In this review, the participation of H2S in many physiological and pathological processes in animals is described, and its importance as a signaling molecule in plant systems is underlined from an evolutionary point of view. H2S quantification methods are summarized and persulfidation is described as the underlying mechanism of action in plants, animals and bacteria. This review aims to highlight the importance of its crosstalk with other signaling molecules and its fine regulation for the proper function of the cell and its survival.EU Marie Skłodowska-Curie 834120Junta de Andalucía US-125578

VPS45 is required for both diffuse and tip growth of Arabidopsis thaliana cells

IntroductionVPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously that Arabidopsis thaliana VPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on the trans-Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth.ResultsAs vps45 knockout mutation is lethal in Arabidopsis, we identified a mutant, vps45-3, with a point mutation in the VPS45 gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However, vps45-3 plants display severe growth defects with significantly reduced organ and cell size, similar to vps45 RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised in vps45-3. Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter in vps45-3 compared to WT.DiscussionWe thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth

RACK1 Associates with Muscarinic Receptors and Regulates M2 Receptor Trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M2 muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M2-immunoprecipitates from M2-expressing cells over those of non-M2 expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M2. We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M2 receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M2 receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M2 receptors to the degradative pathway