Phagophore membrane connections and RAB24 in autophagy

Eukaryotic cells contain membrane-bound organelles to carry out specialized cellular functions. These organelles are inherited in cell division as templates and are augmented by proliferation through production of protein and lipid components by the cell, and the trafficking of these components within the cell. Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce nutrients for energy production, degrade misfolded proteins or damaged whole organelles, and fight against intruding pathogens. The process of autophagy entails the isolation of cargo by a specialized organelle, called the phagophore, which closes to form a sealed double membrane bound autophagosome. This organelle then undergoes maturation by fusion with endosomes and lysosomes to obtain its degradation capacity. Hence, there are many dynamic membrane modifications that need to take place during the autophagic process. The origin of the autophagic limiting membrane, as well as the clearance of the degradative structures, are yet to be defined. This study utilized high resolution electron microscopic methods and three dimensional modeling to reveal nanometer scale interactions of phagophores and autophagosomes with other organelles. Immunolabeling techniques at both light and electron microscopy level were utilized to determine which organelles should be sampled at an ultrastructural level. Direct membrane communication was detected between the phagophore and endoplasmic reticulum (ER), (putative) ER exit sites, mitochondria, the Golgi complex, as well as late endosomes or lysosomes. ER was the most frequent proximal organelle to phagophores and autophagosomes and this suggests an involvement of ER in the nucleation process of phagophores. This study also reveales a role of the small GTP-binding protein RAB24 in the clearance of autophagic structures in cells. Biochemical and microscopic methods in combination showed that RAB24 is needed in the clearance of autophagic structures in nutrient rich conditions i.e. during basal autophagy. RAB24 was confirmed to localize in both of the autophagosome limiting membranes. GTP binding and prenylation of RAB24 were found to be necessary for the targeting of the protein to LC3 positive autophagic structures, whereas tyrosine phosphorylation was less important for this targeting. Electron microscopy revealed that autolysosome-like structures accumulate in cells when RAB24 is silenced, suggesting that it has a role in the clearance of autolysosomes.Aitotumalliset solut sisältävät monia erikoistuneita kalvon ympäröimiä osastoja eli soluelimiä tai organelleja, joissa solujen biologiset prosessit tapahtuvat. Soluelimet periytyvät solunjakautumisessa tytärsoluihin, ja solut voivat säädellä soluelimien kokoa ja lukumäärää tuottamalla niille ominaisia proteiineja ja lipidejä. Rakennusaineet kulkevat soluissa erityisiä kuljetusreittejä pitkin. Yhtä soluille tärkeää hajotusreittiä kutsutaan autofagiaksi, jonka avulla solut voivat kierrättää tarpeettomia ja vaurioituneita osiaan kuten proteiineja tai kokonaisia soluelimiä. Autofagia on myös tärkeä ravinteiden lähde energiantuotantoon, ja sen avulla solut voivat puolustautua solun sisälle päässeitä patogeenejä vastaan. Autofagiassa solu muodostaa erityisen soluelimen, fagoforin, joka eristää hajotettavan materiaalin ja kuroutuu suljetuksi vesikkeliksi. Tämä vesikkeli on kaksoiskalvon ympäröimä ja sitä kutsutaan autofagosomiksi. Autofagosomit kypsyvät yhtymällä endosomeihin ja lysosomeihin ja näin ollen hankkivat hajotukseen tarvittavat entsyymit ja entsyymeille tarvittavat suotuisat olosuhteet. Autofagian toimiminen edellyttää solulta toimivaa kalvokuljetusta ja monia kalvomodifikaatioita. Autofagosomin ympäröivän kalvon alkuperää ei vielä tunneta. Myöskään hajotuksen jälkeisiä tapahtumia, kuten autofagosomien häviämistä solusta, ei tarkoin tunneta. Tässä tutkimuksessa muodostuvaa fagoforia on tarkasteltu erittäin korkean resoluution elektronimikroskooppisilla menetelmillä yhdistettynä kolmiulotteiseen mallinnukseen. Näillä menetelmillä fagoforeja ja autofagosomeja on voitu tarkastella suhteessa muihin soluelimiin nanometrien tarkkuudella. Vasta-aineleimausta käytettiin sekä valo- että elektronimikroskopiassa soluelimien tunnistamiseen. Elektronitomografialla pystyttiin osoittamaan kalvokontakteja fagoforin ja monien muiden soluelinten välillä. Fagofori oli yhteydessä solulimakalvoston eri osiin, mitokondrioihin, Golgin laitteeseen ja myöhäisiin endosomeihin tai lysosomeihin. Muodostuvan fagoforin havaittin olevan myös lähellä lipidipisaraa. Useimmiten fagoforit löytyivät solulimakalvoston välittömästä läheisyydestä, mikä saattaa viitata solulimakalvoston toimintaan fagoforin muodostuksessa, esimerkiksi kalvon luovuttajana. Tässä tutkimuksessa myös osoitettiin, että pieni guaniininukleotidejä sitova proteiini, RAB24, toimii autofagosomien hävittämisessä soluista. RAB24 proteiinin osoitettiin toimivan autofagosomien hävittämisessä soluista ravinteikkaissa olosuhteissa hyväksikäyttäen mikroskooppisia ja biokemiallisia menetelmiä. RAB24:n osoitettiin paikantuvan autofagosomeja ympäröiville kalvoille. Lisäksi RAB24:n kohdentaminen autofagosomien kalvoille oli riippuvaista sen kyvystä sitoa guaniininukleotidejä sekä sen lipidiankkurista. Tyrosiiniaminohappotähteiden fosforylaatio ei ollut välttämätöntä RAB24:n kohdentamiselle autofagosomeihin. Elektronimikroskooppisin menetelmin pystyttiin osoittamaan, että RAB24:n puuttuminen aiheutti autofagosomien kerääntymisen soluihin. Tämä viittaa siihen, että RAB24 tarvitaan autofagosomien tehokkaaseen poistamiseen soluista

Roles for RAB24 in autophagy and disease

Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes in order to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.Peer reviewe

A novel mechanism for regulation of the type I IFN response by herpesvirus deconjugases

Upon infection, viral nucleic acids are recognized by germline-encoded pattern-recognition receptors (PRRs), and cytosolic retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) that initiate signaling pathways resulting in the production of type I IFN and pro-inflammatory cytokines. Binding of RIG-I to viral nucleic acids triggers the formation of the RIG-I signalosome where RIG-I is ubiquitinated by the TRIM25 ligase and, with the help of 14-3-3 scaffolds, further translocated to mitochondrial anti-viral signalling proteins (MAVS). Subsequent ubiquitination-mediated events trigger transcriptional activation of the effectors of innate immunity. We have found a new mechanism by which herpesviruses interfere with this signalling pathway to favour the establishment of latency and promote virus replication. The cysteine protease encoded in the conserved N-terminal domain of the herpesvirus large tegument protein binds to 14-3-3 proteins and forms a tri-molecular complex with TRIM25, promoting the activation and autoubiquitination of the ligase. RIG-I is recruited to the complex but its ubiquitination is drastically reduced, which effectively inactivates downstream signalling and blocks the type I IFN response

RAB24 facilitates clearance of autophagic compartments during basal conditions

Officially accepted for publication August 21, 2015.RAB24 belongs to a family of membrane traffic controlling RAB proteins and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy showed an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicated that this was due to a hindrance in their clearance. Formation of autophagosomes was shown to be unaffected by RAB24 silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 was also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.Peer reviewe

Herpesvirus deconjugases inhibit the IFN response by promoting TRIM25 autoubiquitination and functional inactivation of the RIG-I signalosome

The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. The proteins are expressed during the productive virus cycle and are incorporated into infectious virus particles, being delivered to the target cells upon primary infection. Members of this viral enzyme family were shown to regulate different aspects of the virus life cycle and the innate anti-viral response. However, only few substrates have been identified and the mechanisms of these effects remain largely unknown. In order to gain insights on the substrates and signaling pathways targeted by the viral enzymes, we have used co-immunoprecipitation and mass spectrometry to identify cellular proteins that interact with the Epstein-Barr virus encoded homologue BPLF1. Several members of the 14-3-3-family of scaffold proteins were found amongst the top hits of the BPLF1 interactome, suggesting that, through this interaction, BPLF1 may regulate a variety of cellular signaling pathways. Analysis of the shared protein-interaction network revealed that BPLF1 promotes the assembly of a trimolecular complex including, in addition to 14-3-3, the ubiquitin ligase TRIM25 that participates in the innate immune response via ubiquitination of cytosolic pattern recognition receptor, RIG-I. The involvement of BPLF1 in the regulation of this signaling pathway was confirmed by inhibition of the type-I IFN responses in cells transfected with a catalytically active BPLF1 N-terminal domain or expressing the endogenous protein upon reactivation of the productive virus cycle. We found that the active viral enzyme promotes the dimerization and autoubiquitination of TRIM25. Upon triggering of the IFN response, RIG-I is recruited to the complex but ubiquitination is severely impaired, which functionally inactivates the RIG-I signalosome. The capacity to bind to and functionally inactivate the RIG-I signalosome is shared by the homologues encoded by other human herpesviruses

Ultrastructural relationship of the phagophore with surrounding organelles

<div><p>Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.</p></div

BPLF1 interacting proteins identified by tandem mass spectrometry.

<p><b>A</b>. List of the 22 most abundant BPLF1 interacting proteins identified by mass spectrometry. The values are derived from the mean of two independent experiments. ID = protein ID, MW = molecular weight, Unique Pep = number of unique peptides mapping to the protein, PSM = number of peptide spectrums matched to the protein. INTER = number of annotated interacting proteins found in the BPLF1 interactome. 14-3-3 isoforms: YWHAZ = 14-3-3 ζ/δ, YWHAE = 14-3-3 ε, YWHAG = 14-3-3 γ, YWHAB = 14-3-3 β/α, YWHAH = 14-3-3 η. <b>B</b>. The interaction of BPLF1 with the 14-3-3 isoforms was validated by immunoprecipitation and immunoblotting with the indicated isoform-specific antibodies. <b>C</b>. Circos diagram illustrating the annotated interaction of the 14-3-3 isoforms with other proteins enriched in the BPLF1 interactome. The two ubiquitin ligases found in the shared interactome, CUL1 and TRIM25, are highlighted in red. Color coded annotated functions are shown in the outer ring.</p

The effect of BPLF1 is conserved in homologs encoded by other herpesviruses.

<p><b>A</b>. Functional homologs of BPLF1 encoded by HSV, HCMV and KHSV promote the autoubiquitination of TRIM25. HeLa cells were co-transfected with HA-TRIM25 plasmid and plasmids encoding the indicated catalytically active FLAG-tagged N-terminal domains of herpesvirus deconjugases along with catalytically inactive FLAG-BPLF1C61A. The viral proteins were immunoprecipitated with anti-FLAG-conjugated agarose beads and co-precipitated endogenous TRIM25 was detected in western blots probed with the specific antibody. Mono- and di-ubiquitinated TRIM25 species are indicated by arrows. One representative experiment is shown. <b>B</b>. The herpesvirus deconjugases interact with endogenous 14-3-3 and TRIM25. FLAG-immunoprecipitates were probed with the pan-14-3-3 and TRIM25 antibodies. <b>C</b>. The BPLF1 homologs inhibit the ubiquitination of RIG-I. HA-UbK63 was co-transfected in HeLa cells together with the indicated FLAG-tagged herpesvirus deconjugases. RIG-I ubiquitination was induced by co-transfecting the cells with GST-2CARD. Endogenous RIG-I was immunoprecipitated and ubiquitination was detected by probing immunoblots with the anti-HA antibody. One representative experiment out of 2 is shown.</p