Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.

The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt-Hogg-Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.AP, BKB and RKN were supported by the Myrovlytis Trust. DBA was supported by a NHMRC CJ Martin Fellowship (APP1072476). LHW was supported by Medical Research Council (MRC) studentship, MR/J006580/1 and TPL by University College London. SD was supported by Fondation de France, La Ligue National contre le Cancer (Comité de Paris / Ile-de-France and Comité de l’Oise); TLB and NZ thank the University of Cambridge and The Wellcome Trust for facilities and support.This is the final version of the article. It was first available from Royal Society Publishing via http://dx.doi.org/10.1098/rsob.15017

Evolution of the endomembrane systems of trypanosomatids:conservation and specialisation

Parasite surfaces support multiple functions required for survival within their hosts, and maintenance and functionality of the surface depends on membrane trafficking. To understand the evolutionary history of trypanosomatid trafficking, where multiple lifestyles and mechanisms of host interactions are known, we examined protein families central to defining intracellular compartments and mediating transport, namely Rabs, SNAREs and RabGAPs, across all available Euglenozoa genomes. Bodonids possess a large trafficking repertoire, which is mainly retained by the Trypanosoma cruzi group, with extensive losses in other lineages, particularly African trypanosomes and phytomonads. There are no large-scale expansions or contractions from an inferred ancestor, excluding direct associations between parasitism or host range. However, we observe stepwise secondary losses within Rab and SNARE cohorts (but not RabGAPs). Major changes are associated with endosomal and late exocytic pathways, consistent with the diversity in surface proteomes between trypanosomatids and mechanisms of interaction with the host. Along with the conserved core family proteins, several lineage-specific members of the Rab (but not SNARE) family were found. Significantly, testing predictions of SNARE complex composition by proteomics confirms generalised retention of function across eukaryotes

The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins

Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells

The Yeast Nuclear Pore Complex and Transport Through It

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell’s genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or “Nups”), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC’s role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Studying the mrna-bonding centre of the ribosome on the stages of initiation and elongation of translation

The object of investigation: the prokariotic ribosome E. coli. The work is aimed at studying the location of the matrix RNA on the ribosome in the different initiation comlexes and in the elongation ribosome using the method of the affine modification. The modification of ribosomes by the photoaffine analogs of the mRNA has been performed. Stitches mRNA-16s RNA have been analyzed by the RNA-ase H hydrolisis, the mRNA-proteins using the immunochemical method. The new method intended for the analysis of stitches mRNA-16s RNA has been developed. Specific areas 16s RNA and ribosome proteins, which are drawn nearer with mRNA on the different stages of translation, have been identified. The results have served as the base for the model of space organisation 30s subparticle for reconsiderationAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Le complexe SEA (Structure et Fonction d'un Nouveau Régulateur de la Voie TORC1)

La voie TORC1 joue un rôle majeur dans le contrôle de la croissance cellulaire et de la réponse à divers stress. Le dérèglement de cette voie est constaté dans de nombreux cancers et autres maladies. Au cours de ma thèse, j ai montré que le complexe SEA émerge comme un régulateur central des différentes activités de TORC1. Durant la carence azotée, les délétions des gènes du complexe SEA dans l organisme modèle S.cerevisiae mènent à la délocalisation de la kinase Tor1 vers le cytoplasme, à des défauts d autophagie et à la fragmentation de la vacuole. L inactivation de TORC1 par le traitement avec la rapamycine ou pendant la carence azotée change le niveau d expression des membres du complexe SEA. De plus, le complexe SEA interagit avec la mitochondrie, joue un rôle dans la réponse au stress oxydatif et peut servir de lien moléculaire entre les fonctions mitochondriales et la voie TORC1. Enfin, j ai pu observer que le complexe SEA est impliqué dans les mécanismes de résistance à une drogue souvent utilisée en chimiothérapie, la doxorubicine. Je présente dans mes travaux la première carte d interconnectivité des protéines composant le complexe SEA. Nos données suggèrent que le complexe SEA émerge comme une plateforme qui peut coordonner les activités structurales et enzymatiques nécessaires pour le fonctionnement efficace de la voie de signalisation TORC1.The TORC1 pathway plays a major role in controlling cell growth and response to various stresses. Deregulation of this pathway is found in many cancers and other diseases. In my thesis, I have shown that the SEA complex emerges as a central regulator of the various activities of TORC1. During the nitrogen deficiency, deletions of SEA complex genes in the model organism S.cerevisiae lead to the relocation of Tor1 kinase to the cytoplasm, to defects in autophagy and the fragmentation of the vacuole. Inactivation of TORC1 by treatment with rapamycin or nitrogen starvation changes the level of expression of SEA complex members. Moreover, the SEA complex interacts with mitochondrion, plays a role in oxidative stress response and can serve as a molecular link between mitochondrial functions and TORC1 pathway. Finally, I observed that the SEA complex is involved in the mechanisms of resistance to a drug often used in chemotherapy, the doxorubicin. I present in my work the first interconnectivity map protein of the SEA complex component. Our data suggest that the SEA complex emerges as a platform that can coordinate structural and enzymatic activities necessary for the efficient function of the TORC1 signalling pathway.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF