ORCID Profile
0000-0002-2677-6945
Current Organisations
IT University of Copenhagen
,
Aarhus University
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Publisher: Elsevier BV
Date: 06-2016
Publisher: Wiley
Date: 28-06-2008
DOI: 10.1111/J.1600-0854.2008.00753.X
Abstract: Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with high-pressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.
Publisher: EMBO
Date: 22-03-2017
Publisher: Wiley
Date: 28-12-2014
DOI: 10.1111/TRA.12243
Abstract: Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)-resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM-binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-01-2022
Abstract: Portions of the endoplasmic reticulum (ER) are degraded by autophagy (ER-phagy) in response to starvation or the accumulation of misfolded proteins. We show that ER-phagy requires assembly of actin at sites of contact between the edges of ER sheets and endocytic pits on the plasma membrane. Actin assembly may help to bring an element of the ER carrying the selective autophagy receptor Atg40 into the cell interior, where it associates with Atg11, a scaffold needed to recruit components for autophagosome assembly. Understanding the mechanism by which regions of the ER are selected for degradation and sequestered within autophagosomes may help in the development of novel approaches to treat diseases that result from the accumulation of misfolded proteins within the ER.
Publisher: The Company of Biologists
Date: 15-04-2022
DOI: 10.1242/JCS.259421
Abstract: Lysosomes mediate degradation of macromolecules to their precursors for cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. Chédiak–Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein and an effector of the minor Rab5 isoform Ypt52. Strikingly, bph1Δ mutant cells have lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy-inducing conditions. In agreement with this, proteomic analysis of bph1Δ vacuoles reveals an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1Δ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When Bph1 is lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.
Publisher: Cold Spring Harbor Laboratory
Date: 29-07-2020
DOI: 10.1101/2020.07.28.225391
Abstract: Lipid droplets (LDs) are globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerols and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids harboring a specific set of proteins. Most of these proteins associate with LDs through either an hipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane spanning proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, is due to redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners in cells coexpressing the membrane-anchored perilipin. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of OM14, an outer mitochondrial membrane protein. Expression of OM14-PLIN3 resulted in close apposition of mitochondria and LDs. Taken together, these data indicate that the LD surface constitutes a barrier for ER-localized integral membrane spanning proteins.
Publisher: Springer Science and Business Media LLC
Date: 24-09-2018
DOI: 10.1038/S41419-018-1003-1
Abstract: Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7 - dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.
Publisher: EMBO
Date: 14-07-2015
Publisher: Springer Science and Business Media LLC
Date: 12-07-2021
Publisher: Rockefeller University Press
Date: 15-11-2010
Abstract: Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab guanosine triphosphatases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with the target membranes. Little is known about how tethering factors discriminate between different trafficking pathways, which may converge at the same organelle. In this paper, we describe a phosphorylation-based switch mechanism, which allows the homotypic vacuole fusion protein sorting effector subunit Vps41 to operate in two distinct fusion events, namely endosome–vacuole and AP-3 vesicle–vacuole fusion. Vps41 contains an hipathic lipid-packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is inserted into the lipid bilayer and masks the binding motif for the δ subunit of the AP-3 complex, Apl5, without affecting the Vps41 function in endosome–vacuole fusion. At the much less curved vacuole, the ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the Apl5-binding site is exposed and allows AP-3 vesicles to bind to Vps41, followed by specific fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phosphorylation.
Publisher: Rockefeller University Press
Date: 20-09-2010
Abstract: Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.
Publisher: American Society for Cell Biology (ASCB)
Date: 06-2012
Abstract: In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.
Publisher: Elsevier BV
Date: 11-2001
Publisher: Proceedings of the National Academy of Sciences
Date: 20-07-2020
Abstract: Vps13, a conserved phospholipid transporter, localizes to contact sites between the endoplasmic reticulum (ER) and different organelles. While budding yeast has one VPS13 gene, humans have four. The yeast Vps13 protein is most closely related to VPS13A and VPS13C. VPS13A localizes to ER–mitochondria contact sites, while VPS13C resides at ER–late endosome contact sites. Here we report that the vps13 Δ mutant is defective in the selective autophagy of ER (ER-phagy). In the absence of Vps13, the ER accumulates at late endosomes and fails to be packaged into autophagosomes. These findings highlight the importance of Vps13 function at late endosomes in ER-phagy, and may have implications for understanding the role of VPS13C, which has been linked to early-onset Parkinson’s disease.
Publisher: Informa UK Limited
Date: 10-2007
DOI: 10.1128/MCB.00815-07
Publisher: American Society for Cell Biology (ASCB)
Date: 04-2009
Abstract: Membrane fusion depends on multisubunit tethering factors such as the vacuolar HOPS complex. We previously showed that the vacuolar casein kinase Yck3 regulates vacuole biogenesis via phosphorylation of the HOPS subunit Vps41. Here, we link the identified Vps41 phosphorylation site to HOPS function at the endosome–vacuole fusion site. The nonphosphorylated Vps41 mutant (Vps41 S-A) accumulates together with other HOPS subunits on punctate structures proximal to the vacuole that expand in a class E mutant background and that correspond to in vivo fusion sites. Ultrastructural analysis of this mutant confirmed the presence of tubular endosomal structures close to the vacuole. In contrast, Vps41 with a phosphomimetic mutation (Vps41 S-D) is mislocalized and leads to multilobed vacuoles, indicative of a fusion defect. These two phenotypes can be rescued by overproduction of the vacuolar Rab Ypt7, revealing that both Ypt7 and Yck3-mediated phosphorylation modulate the Vps41 localization to the endosome–vacuole junction. Our data suggest that Vps41 phosphorylation fine-tunes the organization of vacuole fusion sites and provide evidence for a fusion “hot spot” on the vacuole limiting membrane.
Publisher: MDPI AG
Date: 05-09-2017
DOI: 10.3390/V9090251
Publisher: The Company of Biologists
Date: 04-2011
DOI: 10.1242/JCS.072538
Abstract: Many different tissues and cell types exhibit regulated secretion of lipoprotein lipase (LPL). However, the sorting of LPL in the trans Golgi network has not, hitherto, been understood in detail. Here, we characterize the role of SorLA (officially known as SorLA-1 or sortilin-related receptor) in the intracellular trafficking of LPL. We found that LPL bound to SorLA under neutral and acidic conditions, and in cells this binding mainly occurred in vesicular structures. SorLA expression changed the subcellular distribution of LPL so it became more concentrated in endosomes. From the endosomes, LPL was further routed to the lysosomes, which resulted in a degradation of newly synthesized LPL. Consequently, an 80% reduction of LPL activity was observed in cells that expressed SorLA. By analogy, SorLA regulated the vesicle-like localization of LPL in primary neuronal cells. Thus, LPL binds to SorLA in the biosynthetic pathway and is subsequently transported to endosomes. As a result of this SorLA mediated-transport, newly synthesized LPL can be routed into specialized vesicles and eventually sent to degradation, and its activity thereby regulated.
Publisher: Informa UK Limited
Date: 20-07-2018
Publisher: Springer Science and Business Media LLC
Date: 10-2007
DOI: 10.1038/NCB1007-1125
Publisher: American Society for Cell Biology (ASCB)
Date: 15-12-2009
Abstract: Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.
Publisher: EMBO
Date: 13-06-2022
Publisher: Informa UK Limited
Date: 27-03-2007
DOI: 10.4161/AUTO.3608
Abstract: Autophagy can be ided into selective and nonselective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element. Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.
Publisher: Springer Science and Business Media LLC
Date: 24-05-2023
DOI: 10.1038/S41586-023-06090-9
Abstract: Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An ex le of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy) 1 . Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons 2 . Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss 3 , interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1 -deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.
Publisher: Informa UK Limited
Date: 16-11-2010
Abstract: Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.
Publisher: EMBO
Date: 25-08-2020
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-05-2019
Abstract: Muscular dystrophy-associated mutations of TRIM32 impair ULK1-dependent autophagy response during muscle atrophy.
Publisher: eLife Sciences Publications, Ltd
Date: 15-06-2017
DOI: 10.7554/ELIFE.25555
Abstract: The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Publisher: Proceedings of the National Academy of Sciences
Date: 30-01-2001
Abstract: The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell. Rabip4 contains two coiled-coil domains and a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes. The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes. It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1. We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.
Publisher: Frontiers Media SA
Date: 28-04-2022
DOI: 10.3389/FCIMB.2022.855290
Abstract: Latent HIV-1 provirus in infected in iduals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3’ LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo s les. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5’ LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.
Publisher: Informa UK Limited
Date: 09-08-2022
Publisher: Wiley
Date: 21-02-2008
DOI: 10.1111/J.1600-0854.2008.00715.X
Abstract: Proteins carrying EPS15 homology (EH) domains are present from yeast to mammals. The characterized members of this protein family are all involved in intracellular trafficking, typically endocytosis and endocytic recycling. We focused on two members of this family in Saccharomyces cerevisiae Irs4p and Tax4p, whose functions are less well characterized. We show that the deletion of IRS4 altered the function of a neighboring gene, VPS51, involved in endocytic recycling. The irs4Deltatax4Delta cells complemented for the loss of Vps51p (irs4Deltatax4Delta*) display no defects in endocytosis and endosomal recycling, clearly differentiating these two EH proteins from the other protein family members. Because Irs4p is phosphorylated when autophagy is induced, we studied the potential role of these two proteins in this latter process. We observed a loss of viability upon starvation in irs4Deltatax4Delta* cells because of a delay in bulk autophagy. Irs4p and Tax4p are also required for pexophagy but not for the cytoplasm-to-vacuole pathway. In growing cells, Irs4p and Tax4p colocalized to few cytoplasmic puncta distinct from endosomes and Golgi compartments. In conditions inducing autophagy, Irs4p and Tax4p partially localized to the pre-autophagosomal structure (PAS) and are required to efficiently recruit to the PAS Atg17p, a factor modulating the autophagic response. We propose that Irs4p and Tax4p are two redundant modulators of the autophagic processes acting upstream from Atg17p, possibly in the signaling events leading to the activation of the autophagic machinery in response to starvation.
Publisher: Rockefeller University Press
Date: 29-07-2021
Abstract: Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.
Publisher: Springer Science and Business Media LLC
Date: 10-12-2021
DOI: 10.1038/S41467-021-27420-3
Abstract: Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-07-2019
Abstract: In eukaryotic cells, about one-third of all proteins are targeted to the endoplasmic reticulum (ER), which serves as a hub for secretory protein traffic and quality control. Cui et al. studied a protein known as Lst1 in yeast and SEC24C in mammalian cells that is involved in loading secretory cargo into vesicles that are delivered to the Golgi complex. In response to stress caused by starvation or misfolded aggregate-prone secretory proteins, Lst1 acted to promote an additional function—ER-phagy. Together with autophagy receptors on the ER, Lst1 targeted ER domains for degradation to avert protein aggregation, thus preserving cellular health. Science , this issue p. 53
Publisher: Springer Science and Business Media LLC
Date: 09-09-2020
DOI: 10.1007/S12529-020-09928-Y
Abstract: In the past decades, behavioral medicine has attained global recognition. Due to its global reach, a critical need has emerged to consider whether the original definition of behavioral medicine is still valid, comprehensive, and inclusive, and to reconsider the main tasks and goals of the International Society of Behavioral Medicine (ISBM), as the umbrella organization in the field. The purpose of the present study was to (i) update the definition and scope of behavioral medicine and its defining characteristics and (ii) develop a proposal on ISBM’s main tasks and goals. Our study used the Delphi method. A core group prepared a discussion paper. An international Delphi panel rated questions and provided comments. The panel intended to reach an a priori defined level of consensus (i.e., 70%). The international panel reached consensus on an updated definition and scope of behavioral medicine as a field of research and practice that builds on collaboration among multiple disciplines. These disciplines are concerned with development and application of behavioral and biomedical evidence across the disease continuum in clinical and public health domains. Consensus was reached on a proposal for ISBM’s main tasks and goals focused on supporting communication and collaboration across disciplines and participating organizations stimulating research, education, and practice and supporting in iduals and organizations in the field. The consensus on definition and scope of behavioral medicine and ISBM’s tasks and goals provides a foundational step toward achieving these goals.
Publisher: Cold Spring Harbor Laboratory
Date: 08-02-2021
DOI: 10.1101/2021.02.08.429707
Abstract: Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes including yeast. PC has been assigned a multitude of functions in addition to that of building block of the lipid bilayer. Here we show that PC is evolvable essential in yeast by isolating suppressor mutants devoid of PC that exhibit robust growth. The requirement for PC is suppressed by monosomy of chromosome XV, or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis differently, both decrease Acc1 activity thereby reducing the average acyl chain length. Accordingly, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, up-regulation of lipid synthesis is instrumental to accomplish feed-back inhibition of Acc1 by acyl-CoA produced by the fatty acid synthase (FAS). The results show that yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS, and indicate that PC evolved by benefitting the maintenance of membrane fluidity.
Publisher: Cold Spring Harbor Laboratory
Date: 14-08-2019
DOI: 10.1101/733931
Abstract: Lipid droplets are unique and nearly ubiquitous organelles that store neutral lipids in a hydrophobic core, surrounded by a monolayer of phospholipids. The primary neutral lipids are triacylglycerols and steryl esters. It is not known whether other classes of neutral lipids can form lipid droplets by themselves. Here we show that production of retinyl esters by lecithin:retinol acyl transferase (LRAT) in yeast cells, incapable of producing triacylglycerols and steryl esters, causes the formation of lipid droplets. By electron microscopy, these lipid droplets are morphologically indistinguishable from those in wild-type cells. In silico and in vitro experiments confirmed the propensity of retinyl esters to segregate from membranes and to form lipid droplets. The hydrophobic N-terminus of LRAT displays preferential interactions with retinyl esters in membranes and promotes the formation of large retinyl ester-containing lipid droplets in mammalian cells. Our combined data indicate that the molecular design of LRAT is optimally suited to allow the formation of characteristic large lipid droplets in retinyl ester-storing cells.
Publisher: Wiley
Date: 28-01-2003
DOI: 10.1034/J.1600-0854.2003.40205.X
Abstract: The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4. We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS-positive vesicles. However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.
Publisher: Shared Science Publishers OG
Date: 02-11-2015
Publisher: EMBO
Date: 14-09-2021
Publisher: Faculty Opinions Ltd
Date: 12-2011
DOI: 10.3410/B3-25
Publisher: Informa UK Limited
Date: 13-05-2012
DOI: 10.4161/AUTO.19385
Publisher: American Society for Cell Biology (ASCB)
Date: 05-2018
Abstract: Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar serine carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.
Publisher: Elsevier BV
Date: 09-2019
Publisher: Springer Science and Business Media LLC
Date: 10-08-2022
DOI: 10.1038/S41467-022-32487-7
Abstract: The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1 .
Publisher: Wiley
Date: 06-11-2007
DOI: 10.1111/J.1600-0854.2007.00674.X
Abstract: Autophagy is a catabolic process conserved among all eukaryotes essential for the cellular and organismal homeostasis. One of the principal roles of this pathway is to maintain an accurate balance between synthesis, degradation and subsequent recycling of cellular components. Under certain conditions, however, cells are also able to modulate autophagy and specifically remove a number of structures that are potentially harmful. Aberrant protein aggregates, damaged organelles or pathogens can be selectively incorporated into large double-membrane vesicles called autophagosomes to be delivered into lysosomes for destruction. This ability to eliminate specific structures is exploited by the cells in several physiological processes as well as in multiple pathological situations, making autophagy a precious multitask cellular degradative pathway. In this review, we will first examine what is known about the basic mechanisms of autophagy and then discuss in a second part the nature of the cargoes that are selectively sequestered into autophagosomes, what provides the specificity and the possible implications of selective types of autophagy in human pathologies.
Publisher: Springer Science and Business Media LLC
Date: 15-06-2021
DOI: 10.1186/S40035-021-00243-4
Abstract: Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2 . To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35 . These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches. Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired. Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.
Publisher: Elsevier BV
Date: 05-2023
Publisher: Rockefeller University Press
Date: 29-11-2017
Abstract: Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus–vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.
Publisher: American Physical Society (APS)
Date: 29-08-2023
Publisher: Informa UK Limited
Date: 21-06-2020
Publisher: The Company of Biologists
Date: 15-07-2011
DOI: 10.1242/JCS.076836
Abstract: Cells store metabolic energy in the form of neutral lipids that are deposited within lipid droplets (LDs). In this study, we examine the biogenesis of LDs and the transport of integral membrane proteins from the endoplasmic reticulum (ER) to newly formed LDs. In cells that lack LDs, otherwise LD-localized membrane proteins are homogenously distributed in the ER membrane. Under these conditions, transcriptional induction of a diacylglycerol acyltransferase that catalyzes the formation of the storage lipid triacylglycerol (TAG), Lro1, is sufficient to drive LD formation. Newly formed LDs originate from the ER membrane where they become decorated by marker proteins. Induction of LDs by expression of the second TAG-synthesizing integral membrane protein, Dga1, reveals that Dga1 itself moves from the ER membrane to concentrate on LDs. Photobleaching experiments (FRAP) indicate that relocation of membrane proteins from the ER to LDs is independent of temperature and energy, and thus not mediated by classical vesicular transport routes. LD-localized membrane proteins are homogenously distributed at the perimeter of LDs, they are free to move over the LD surface and can even relocate back into the ER, indicating that they are not restricted to specialized sites on LDs. These observations indicate that LDs are functionally connected to the ER membrane and that this connection allows the efficient partitioning of membrane proteins between the two compartments.
Publisher: The Company of Biologists
Date: 2019
DOI: 10.1242/JCS.235002
Abstract: Autophagy is initiated by the formation of phagophore assembly sites (PAS), the precursors of autophagosomes. In mammals, PAS form throughout the cytosol in specialized subdomains of the endoplasmic reticulum (ER). In yeast, the PAS is also generated close to the ER, but always in the vicinity of the vacuole. How the PAS is anchored to the vacuole and the functional significance of this localization are unknown. Here, we investigated the role of the PAS-vacuole connection for bulk autophagy in yeast. We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13. S. cerevisiae lacking Vac8 show inefficient autophagosome-vacuole fusion, and form fewer and smaller autophagosomes that often localize away from the vacuole. Thus, the stable PAS-vacuole connection established by Vac8 creates a confined space for autophagosome biogenesis between the ER and the vacuole and allows spatial coordination of autophagosome formation and autophagosome-vacuole fusion. These findings reveal that the spatial regulation of autophagosome formation at the vacuole is required for efficient bulk autophagy.
Publisher: Informa UK Limited
Date: 12-1996
Publisher: Springer Science and Business Media LLC
Date: 02-05-2018
DOI: 10.1038/S41467-018-03957-8
Abstract: The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in β-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.
Publisher: Informa UK Limited
Date: 19-09-2019
Publisher: Cold Spring Harbor Laboratory
Date: 27-10-2021
DOI: 10.1101/2021.10.26.465890
Abstract: Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage/crossing of biomolecules. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separate 1,2 and aggregate when isolated 3 . It has remained largely unclear how FG-Nups are protected from making inappropriate interactions during NPC biogenesis. We found that DNAJB6, a molecular chaperone of the heat shock protein network, formed foci next to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Reversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and they could be identified as herniations at the nuclear envelope (NE). Immunoelectron tomography showed that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Interestingly, loss of DNAJB6 results in annulate lamellae, which are structures containing partly assembled NPCs associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG-region of several FG-Nups in cells and in vitro . Together, our data show that DNAJB6 provides quality control during NPC biogenesis and is the first molecular chaperone that is involved in the surveillance of native intrinsically disordered proteins, including FG-Nups.
Publisher: The Company of Biologists
Date: 24-05-2021
DOI: 10.1242/JCS.256206
Abstract: Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an hipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14–PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER–LD junction constitutes a barrier for ER-resident integral membrane proteins. This article has an associated First Person interview with the first author of the paper.
Publisher: MyJove Corporation
Date: 14-07-2014
DOI: 10.3791/51752
Publisher: Rockefeller University Press
Date: 29-06-2022
Abstract: Membrane contact sites are specialized platforms formed between most organelles that enable them to exchange metabolites and influence the dynamics of each other. The yeast vacuole is a degradative organelle equivalent to the lysosome in higher eukaryotes with important roles in ion homeostasis and metabolism. Using a high-content microscopy screen, we identified Ymr160w (Cvm1, for contact of the vacuole membrane 1) as a novel component of three different contact sites of the vacuole: with the nuclear endoplasmic reticulum, the mitochondria, and the peroxisomes. At the vacuole–mitochondria contact site, Cvm1 acts as a tether independently of previously known tethers. We show that changes in Cvm1 levels affect sphingolipid homeostasis, altering the levels of multiple sphingolipid classes and the response of sphingolipid-sensing signaling pathways. Furthermore, the contact sites formed by Cvm1 are induced upon a decrease in sphingolipid levels. Altogether, our work identifies a novel protein that forms multiple contact sites and supports a role of lysosomal contacts in sphingolipid homeostasis.
Publisher: Cold Spring Harbor Laboratory
Date: 07-05-2019
DOI: 10.1101/629972
Abstract: Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria employ lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa , which has developed erse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, that extend beyond its role as an adhesion molecule. We found that LecB associates with several growth factor receptors and d ens their signalling pathways, leading to the arrest of cell cycle. Additionally, we describe a novel LecB-triggered mechanism to downregulate host cell receptors by showing that LecB leads to insulin-like growth factor receptor 1 internalisation, without receptor activation, and subsequent missorting towards intracellular endosomal compartments. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.
Publisher: Informa UK Limited
Date: 28-10-2020
Publisher: The Company of Biologists
Date: 04-2006
DOI: 10.1242/JCS.02850
Abstract: Insulin regulates glucose uptake in the adipocytes by modulating Glut 4 localization, a traffic pathway involving the endocytic small GTPases Rab4, Rab5, and RabThe expression of the Rab4 effector Rabip4 leads to a 30% increase in glucose uptake and Glut 4 translocation in the presence of insulin, without modifications in the basal condition. This effect was not due to modifications of Glut 4 expression or insulin signaling, suggesting that Rabip4 controls Glut 4 trafficking. We present evidence that Rabip4 defines a subdomain of early endosomes and that Rabip4 is redistributed to the plasma membrane by insulin. Rabip4 is mostly absent from structures positive for early endosome antigen 1, Rab11 or transferrin receptors and from Glut 4 sequestration compartments. However, Rabip4 vesicles can be reached by internalized transferrin and Glut 4. Thus, Rabip4 probably defines an endocytic sorting platform for Glut 4 towards its sequestration pool. The expression of a form of Rabip4 unable to bind Rab4 does not modify basal and insulin-induced glucose transport. However, it induces an increase in the amount of Glut 4 at the plasma membrane and perturbs Glut 4 traffic from endosomes towards its sequestration compartments. These observations suggest that the uncoupling between Rabip4 and Rab4 induces the insertion of Glut 4 molecules that are unable to transport glucose into the plasma membrane.
Publisher: Springer Science and Business Media LLC
Date: 27-10-2022
DOI: 10.1038/S41556-022-01010-X
Abstract: Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2015
DOI: 10.1038/NATURE14498
Abstract: The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.
Publisher: Informa UK Limited
Date: 02-01-2016
Publisher: American Society for Cell Biology (ASCB)
Date: 07-2013
Abstract: Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail–dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p–dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail–dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.
Publisher: EMBO
Date: 27-10-2022
Abstract: Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single‐membrane cisternae called phagophores. While the role of phosphatidylinositol 3‐phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well‐studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP‐DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline‐dependent recovery of de novo PC biosynthesis via the CDP‐choline pathway restores autophagosome formation and autophagic flux in PC‐deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.
Publisher: Informa UK Limited
Date: 26-11-2021
Publisher: Elsevier
Date: 2005
Publisher: Elsevier BV
Date: 2020
Location: France
No related grants have been discovered for Muriel Mari.