A stable microtubule bundle formed through an orchestrated multistep process controls quiescence exit.

Cells fine-tune microtubule assembly in both space and time to give rise to distinct edifices with specific cellular functions. In proliferating cells, microtubules are highly dynamics, and proliferation cessation often leads to their stabilization. One of the most stable microtubule structures identified to date is the nuclear bundle assembled in quiescent yeast. In this article, we characterize the original multistep process driving the assembly of this structure. This Aurora B-dependent mechanism follows a precise temporality that relies on the sequential actions of kinesin-14, kinesin-5, and involves both microtubule-kinetochore and kinetochore-kinetochore interactions. Upon quiescence exit, the microtubule bundle is disassembled via a cooperative process involving kinesin-8 and its full disassembly is required prior to cells re-entry into proliferation. Overall, our study provides the first description, at the molecular scale, of the entire life cycle of a stable microtubule structure in vivo and sheds light on its physiological function.

Post-ER degradation of misfolded GPI-anchored proteins is linked with microautophagy.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are membrane-conjugated cell-surface proteins with diverse structural, developmental, and signaling functions and clinical relevance. Typically, after biosynthesis and attachment to the preassembled GPI anchor, GPI-APs rapidly leave the endoplasmic reticulum (ER) and rely on post-ER quality control. Terminally misfolded GPI-APs end up inside the vacuole/lysosome for degradation, but their trafficking itinerary to this organelle and the processes linked to their uptake by the vacuole/lysosome remain uncharacterized. In a yeast mutant that is lacking Pep4, a key vacuolar protease, several misfolded model GPI-APs accumulated in the vacuolar membrane. In the same mutant, macroautophagy and the multi-vesicular body (MVB) pathway were intact, hinting at a hitherto-unknown trafficking pathway for the degradation of misfolded GPI-APs. To unravel it, we used a genome-wide screen coupled to high-throughput fluorescence microscopy and followed the fate of the misfolded GPI-AP: Gas1∗. We found that components of the early secretory and endocytic pathways are involved in its targeting to the vacuole and that vacuolar transporter chaperones (VTCs), with roles in microautophagy, negatively affect the vacuolar uptake of Gas1∗. In support, we demonstrate that Gas1∗ internalizes from vacuolar membranes into membrane-bound intravacuolar vesicles prior to degradation. Our data link post-ER degradation with microautophagy.

Unbiased yeast screens identify cellular pathways affected in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disease caused by mutations in either the NPC1 or NPC2 genes. Mutations in the NPC1 gene lead to the majority of clinical cases (95%); however, the function of NPC1 remains unknown. To gain further insights into the biology of NPC1, we took advantage of the homology between the human NPC1 protein and its yeast orthologue, Niemann-Pick C-related protein 1 (Ncr1). We recreated the NCR1 mutant in yeast and performed screens to identify compensatory or redundant pathways that may be involved in NPC pathology, as well as proteins that were mislocalized in NCR1-deficient yeast. We also identified binding partners of the yeast Ncr1 orthologue. These screens identified several processes and pathways that may contribute to NPC pathogenesis. These included alterations in mitochondrial function, cytoskeleton organization, metal ion homeostasis, lipid trafficking, calcium signalling, and nutrient sensing. The mitochondrial and cytoskeletal abnormalities were validated in patient cells carrying mutations in NPC1, confirming their dysfunction in NPC disease.

Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation.

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization.

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling.

Disease-causing mutations in subunits of OXPHOS complex I affect certain physical interactions.

Mitochondrial complex I (CI) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human CI structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 CI subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors to serve as both 'prey' and 'bait' in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS CI mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus offering a possible mechanistic explanation for the underlying pathogenicity.

Iron affects Ire1 clustering propensity and the amplitude of endoplasmic reticulum stress signaling.

The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1-mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis.

Cellular Consequences of Diminished Protein O-Mannosyltransferase Activity in Baker's Yeast.

O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in N-glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall ß-1,6-glucan showed impaired growth when O-mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in N-glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of N-glycosylation promoted sensitivity to the drug. Interestingly, even though S. cerevisiae Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.

Water-Transfer Slows Aging in Saccharomyces cerevisiae.

Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process.

A defect in the RNA-processing protein HNRPDL causes limb-girdle muscular dystrophy 1G (LGMD1G).

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies.