Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8481228 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.
| S-EPMC7690952 | biostudies-literature
Project description:BackgroundMembrane trafficking is a defining feature of eukaryotic cells, and is essential for the maintenance of organelle homeostasis and identity. We previously identified Scy1-like 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, as a high-affinity binding partner of COPI coats. COPI-coated vesicles control Golgi to endoplasmic reticulum trafficking and we observed that disruption of Scyl1 function leads to a decrease in trafficking of the KDEL receptor via the COPI pathway. We reasoned that if Scyl1 plays a major role in COPI trafficking its disruption could influence Golgi homeostasis.Methodology/principal findingsWe performed Scyl1 knock down in cultured cells using previously established methods and observed an alteration in Golgi morphology. Both the surface area and volume of the Golgi is increased in Scyl1-depleted cells, but the continuity and polarity of the organelle is unperturbed. At the ultrastructural level we observe a decrease in the orderly structure of the Golgi with an increase in cisternal luminal width, while the number of Golgi cisternae remains unchanged. The golgin family of proteins forms a detergent resistant network that controls Golgi homeostasis. Disruption of this protein network by knock down of the golgin p115 disrupts the Golgi localization of Scyl1. Moreover, we find that Scyl1 interacts with 58K/formiminotransferase cyclodeaminase (FTCD), a protein that is tightly associated with the cis face of the Golgi.Conclusions/significanceOur results place Scyl1 at an interface between the golgin network and COPI trafficking and demonstrate that Scyl1 is required for the maintenance of Golgi morphology. Coupled with the observation from others that Scyl1 is the gene product responsible for the neurodegenerative mouse model mdf, our results additionally implicate the regulation of COPI trafficking and Golgi homeostasis in neurodegeneration.
| S-EPMC2832016 | biostudies-literature
Project description:Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.
| S-EPMC3605407 | biostudies-literature
Project description:Depletion of EHD3 affects sorting in endosomes by altering the kinetics and route of receptor recycling to the plasma membrane. Here we demonstrate that siRNA knockdown of EHD3, or its interaction partner rabenosyn-5, causes redistribution of sorting nexin 1 (SNX1) to enlarged early endosomes and disrupts transport of internalized Shiga toxin B subunit (STxB) to the Golgi. Moreover, under these conditions, Golgi morphology appears as a series of highly dispersed and fragmented stacks that maintain characteristics of cis-, medial- and trans-Golgi membranes. Although Arf1 still assembled onto these dispersed Golgi membranes, the level of AP-1 gamma-adaptin recruited to the Golgi was diminished. Whereas VSV-G-secretion from the dispersed Golgi remained largely unaffected, the distribution of mannose 6-phosphate receptor (M6PR) was altered: it remained in peripheral endosomes and did not return to the Golgi. Cathepsin D, a hydrolase that is normally transported to lysosomes via an M6PR-dependent pathway, remained trapped at the Golgi. Our findings support a role for EHD3 in regulating endosome-to-Golgi transport, and as a consequence, lysosomal biosynthetic, but not secretory, transport pathways are also affected. These data also suggest that impaired endosome-to-Golgi transport and the resulting lack of recruitment of AP-1 gamma-adaptin to Golgi membranes affect Golgi morphology.
| S-EPMC2724733 | biostudies-literature
Project description:Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, "de-envelopment" and "re-envelopment" is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the "primary" envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols. Results: The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the cis face. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.
| S-EPMC6080407 | biostudies-literature
Project description:Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.
| S-EPMC2096593 | biostudies-literature
Project description:Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.
| S-EPMC8021152 | biostudies-literature
Project description:Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus-pituitary-somatotropic hypoplasia. The unbiased smpd3-/- mouse mutant and derived smpd3-/- primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3-/- mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.
| S-EPMC5260872 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2013-09-07 | GSE50680 | GEO
Project description:Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46-60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.
| S-EPMC2832797 | biostudies-literature