Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves

TFK1, a basal body transition fibre protein that is essential for cytokinesis in Trypanosoma brucei

In Trypanosoma brucei, transition fibres (TFs) form a nine-bladed pattern-like structure connecting the base of the flagellum to the flagellar pocket membrane. Despite the characterization of two TF proteins, CEP164C and T. brucei (Tb)RP2, little is known about the organization of these fibres. Here, we report the identification and characterization of the first kinetoplastid-specific TF protein, named TFK1 (Tb927.6.1180). Bioinformatics and functional domain analysis identified three distinct domains in TFK1 – an N-terminal domain of an unpredicted function, a coiled-coil domain involved in TFK1–TFK1 interaction and a C-terminal intrinsically disordered region potentially involved in protein interaction. Cellular immunolocalization showed that TFK1 is a newly identified basal body maturation marker. Furthermore, using ultrastructure expansion and immuno-electron microscopies we localized CEP164C and TbRP2 at the TF, and TFK1 on the distal appendage matrix of the TF. Importantly, RNAi-mediated knockdown of TFK1 in bloodstream form cells induced misplacement of basal bodies, a defect in the furrow or fold generation, and eventually cell death. We hypothesize that TFK1 is a basal body positioning-specific actor and a key regulator of cytokinesis in the bloodstream form Trypanosoma brucei

An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence.

International audienceThe microtubule cytoskeleton is a highly dynamic network. In dividing cells, its complex architecture not only influences cell shape and movement but is also crucial for chromosome segregation. Curiously, nothing is known about the behavior of this cellular machinery in quiescent cells. Here we show that, upon quiescence entry, the Saccharomyces cerevisiae microtubule cytoskeleton is drastically remodeled. Indeed, while cytoplasmic microtubules vanish, the spindle pole body (SPB) assembles a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced. Kinetochores remain attached to microtubule tips but lose SPB clustering and distribute along the microtubule array, leading to a large reorganization of the nucleus. When cells exit quiescence, the nuclear microtubule array slowly depolymerizes and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration. Finally, mutants that do not assemble a nuclear array of microtubules are impaired for both quiescence survival and exit

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in β-sheets and forms quickly large aggregates (> μm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein-protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes

Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro

Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties. (C) 2016 Elsevier B.V. All rights reserved

A stable microtubule array drives fission yeast polarity reestablishment upon quiescence exit

Cells perpetually face the decision to proliferate or to stay quiescent. Here we show that upon quiescence establishment, Schizosaccharomyces pombe cells drastically rearrange both their actin and microtubule (MT) cytoskeletons and lose their polarity. Indeed, while polarity markers are lost from cell extremities, actin patches and cables are reorganized into actin bodies, which are stable actin filament containing structures. Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle. We have identified proteins involved in this process and propose a molecular model for Q-MT bundle formation. Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state. Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation

(A) Colocalization of 19S RP and 20S CP proteasome subunits in quiescent yeast cells

Cells coexpressing the indicated fusion proteins were imaged after 4 d of growth in YPDA at 30°C. GFP fluorescence is green and RFP fluorescence is red. Images are single focal planes. (B) Cells expressing Scl1p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. For each time point, cells displaying both the green and the red fluorescence were scored as indicated in the legend. For each time point, > 200 (two experiments; error bars show SD). Typical colocalization images corresponding to each type of proteasome localization pattern are shown on the right. Images are maximal projection of z stacks. Bars, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Reversible cytoplasmic localization of the proteasome in quiescent yeast cells"</p><p></p><p>The Journal of Cell Biology 2008;181(5):737-745.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396804.</p><p></p

The mitochondrial phosphatidylserine decarboxylase Psd1 is involved in nitrogen starvation-induced mitophagy in yeast

International audienceMitophagy, the selective degradation of mitochondria by autophagy, is a central process that is essential for the maintenance of cell homeostasis. It is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators to perform. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with the ubiquitin-like Atg8 protein, promoting the recruitment of mitochondria to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to a phosphatidylethanolamine tail. In Saccharomyces cerevisiae, several phosphatidylethanolamine synthesis pathways have been characterized, but their contribution to autophagy and mitophagy are unknown. Through different approaches, we show that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only after nitrogen starvation, whereas Psd2, which is located in vacuole, Golgi and endosome membranes, is required preferentially for mitophagy induction in the stationary phase of growth but also to a lesser extent for nitrogen starvation-induced mitophagy. Our results suggest that the mitophagy defect observed in Δpsd1 yeast cells after nitrogen starvation may be due to a failure of Atg8 recruitment to mitochondria.This article has an associated First Person interview with the first author of the paper

(A) PSGs, as revealed by Pre6p-GFP fluorescence, were observed in cells grown for 4 d in different carbon sources containing rich media and in diploid cells grown in YPDA

For each condition > 200 (two experiments; error bars show SD). Typical images (maximal projection of z stacks) of cells displaying PSGs (Pre6p-GFP) are shown on the right. (B) Cells expressing Pre6p-GFP were grown in YPDA medium at 30°C, fixed, and stained with Alexa Fluor phalloidin to reveal F-actin–containing structures. Actively proliferating cells (left) displayed actin patches and cables (red) and a typical Pre6p-GFP nuclear localization. Quiescent cells displayed PSGs (green fluorescence) that did not colocalize with actin bodies (red). (C) Cells coexpressing Dcp2p-GFP and Pup1p-RFP were grown in YPDA medium at 30°C. Actively proliferating yeast cells displayed a typical Pup1p-RFP nuclear localization and small and discrete Dcp2p-GFP dots. In quiescent cells, Dcp2p-GFP localized in P-bodies that did not colocalize with PSGs. Images in B and C are single focal planes. Bars, 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Reversible cytoplasmic localization of the proteasome in quiescent yeast cells"</p><p></p><p>The Journal of Cell Biology 2008;181(5):737-745.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396804.</p><p></p