Degradative Compartments Research Articles

Abstract 21: Sortilin Is a Novel Mediator of Cellular Low-Density Lipoprotein Uptake

Using genome-wide association studies and follow-up functional studies, we recently identified the SORT1 gene as a novel regulator of blood low-density-lipoprotein cholesterol (LDL-C) levels. SORT1 encodes sortilin, also known as neurotensin receptor 3, a protein that functions as a multiligand sorting receptor. Sortilin localizes to various intracellular compartments including the Golgi apparatus and the plasma membrane, and it has roles in both endocytosis and intracellular trafficking of other proteins. To test whether sortilin regulates blood LDL-C levels by influencing cellular uptake of LDL particles, we used a lentiviral delivery system to stably overexpress the wild-type protein or either of two trafficking mutants_the first, designated LAYA, traffics to the plasma membrane but cannot undergo endocytosis, and the second, designated STOP, remains trapped in the Golgi apparatus. When overexpressed in cultured human hepatocellular carcinoma cells, we found that wild-type sortilin dramatically increases LDL uptake into the endolysosomal compartment for degradation, and the LAYA mutant robustly traps LDL particles in the plasma membrane, whereas the STOP mutant does not affect LDL uptake. We observed similar effects in skin fibroblasts taken from a patient with familial hypercholesterolemia_and thus are deficient in LDL receptor function_suggesting that sortilin can facilitate LDL uptake independently of the LDL receptor. As a complementary approach, we have generated human SORT1 knockout cells, using the novel TAL effector nuclease (TALEN) technology, to introduce frameshift mutations into the coding sequence and thereby disrupt the SORT1 alleles. Besides providing a proof-of-principle that we can for the first time rapidly and efficiently generate gene knockouts in human cells, the SORT1 knockout cells are allowing us to assess the effects of sortilin deficiency on LDL uptake.

Abstract 5: Sortilin Regulates Hepatic VLDL Secretion and LDL Uptake in a Lysosome-Dependent Manner

Sortilin, the protein product of the SORT1 gene, is a multi-ligand sorting receptor involved in Golgi to lysosome and plasma membrane to lysosome protein trafficking. Genome wide association studies for lipid traits have identified the 1p13 locus harboring the SORT1 gene as strongly associated both with plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) risk in humans. Adeno-associated virus (AAV)-mediated hepatic sortilin overexpression in LDL receptor deficient mice reduced plasma cholesterol by 30% at two weeks ( n = 6 mice per group, P = 0.02), with a concomitant reduction in LDL-C. In vivo VLDL production studies demonstrated a 50% reduction in the VLDL triglyceride secretion rate ( P = 0.007) and a 50% reduction in apoB secretion ( P = 0.02) with sortilin overexpression. In vivo LDL turnover studies demonstrated a 3-fold increase in the LDL fractional catabolic rate (FCR) with sortilin overexpression ( n = 6 mice per group, P = 0.00002). Sortilin deficiency both alone and on an LDL receptor deficient background led to a 40% and 50% reduction in FCR ( n = 6 mice per group, P = 0.002 and P = 0.01). The effect of sortilin on both VLDL secretion and LDL turnover is dependent on the ability of sortilin to traffic to the lysosome, as sortilin mutants that cannot traffic to the lysosome do not affect VLDL secretion or LDL uptake in vivo or in vitro . Surface plasmon reasonance demonstrated a high affinity interaction between sortilin and the apoB in LDL particles at physiological pH with a K d of ∼2 nM, and this affinity virtually disappears at the acidic lysosomal pH. In sum, these data are consistent with a model in which sortilin binds apoB-containing lipoprotein particles in the Golgi apparatus and at the plasma membrane and traffics them to the endolysosomal compartment for degradation, thereby reducing VLDL secretion and facilitating LDL uptake, explaining the strong association of hepatic sortilin overexpression in humans with reduced plasma cholesterol.

A Model System to Investigate Antibody Bipolar Bridging Mediated by gE‐gI, a Herpes Virus Fc Receptor

The Herpes Simplex Virus 1 (HSV‐1) expresses two membrane glycoproteins (gE and gI) that bind the Fc region of host immunoglobulin G (IgG), which allows HSV‐infected cells to escape recognition by Fc‐dependent immune cells. HSV‐1 gE‐gI can further undermine the immune system by participating in antibody bipolar bridging (ABB), a process by which the antigen‐binding fragments of the IgG bind a viral antigen while the Fc binds to gE‐gI. The fate of the gE‐gI/IgG/viral antigen ABB complex is still unknown. In this study, we engineered an in vitro ABB system to determine whether the gE‐gI/IgG/viral antigen complex is internalized and targeted for degradation in host cells. This system involves HeLa cells transiently expressing HSV‐1 gE‐gI and HSV‐1 gD; and two forms of the monoclonal anti‐gD antibody: one in which the Fabs were fused to a human IgG1 Fc (hIgG), which can bind to gE‐gI via its Fc and to gD via its Fabs, and one in which the Fabs were fused to a mouse IgG2a Fc (mIgG), which can bind to gD but not to gE‐gI. A hIgG against an irrelevant HIV‐1 antigen was used as a control. Time‐lapse confocal imaging showed that gE‐gI internalized hIgG/gD but not mIgG/gD; internalized IgG and gD were targeted into degradative compartments. These results suggest that gE‐gI plays an active role in clearing the infected cell surface of both host IgG and viral antigens, providing HSV‐1 with a mechanism to evade the immune responses.

The intriguing life of autophagosomes.

Autophagosomes are double-membrane vesicles characteristic of macroautophagy, a degradative pathway for cytoplasmic material and organelles terminating in the lysosomal or vacuole compartment for mammals and yeast, respectively. This highly dynamic, multi-step process requires significant membrane reorganization events at different stages of the macroautophagic process. Such events include exchange and flow of lipids and proteins between membranes and vesicles (e.g., during initiation and growth of the phagophore), vesicular positioning and trafficking within the cell (e.g., autophagosome location and movement) and fusion of autophagosomes with the boundary membranes of the degradative compartment. Here, we review current knowledge on the contribution of different organelles to the formation of autophagosomes, their trafficking and fate within the cell. We will consider some of the unresolved questions related to the molecular mechanisms that regulate the “life and death” of the autophagosome.

Assessing compensation for loss of vacuolar function inSaccharomyces cerevisiae

We analyzed how Saccharomyces cerevisiae cells compensate for the lack of a functional vacuole, an acidic membrane-bound degradative and ion storage compartment. We hypothesized that cells lacking a functional vacuole would compensate for the loss of the functions of the vacuole by altering gene expression and (or) metabolic flux. We used gene expression profiling and Biolog phenotype microarray analysis to determine the compensatory mechanisms of cells lacking vacuolar function. In steady state, vps33 and vps41 cells changed the transcriptional profile of some genes, but no complete pathways were upregulated or downregulated. We treated vps41 cells with calcium to tease out cellular compensation for loss of vacuole function under ionic stress; however, changes in gene expression were not utilized to compensate for loss of vacuole function under stress either, as genes whose transcriptional profiles were changed did not function together in any one cellular process. Phenotype microarray analysis indicated that logarithmically growing vps33 or vps41 cells did not seem to compensate for loss of vacuolar function but instead demonstrated additional pleiotropic phenotypes due to the function of the vacuole. Under rich media conditions, yeast utilize the vacuole to regulate stress, ion response, and peptide degradation. However, loss of the vacuole does not lead to observable compensation mechanisms.

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.

A Case of Hermansky Pudlak Syndrome Type 2

Abstract 4919Hermansky Pudlak Syndrome (HPS) is a genetically heterogeneous group of rare disorders that are classified as ‘lysosome biogenesis disorders' and are characterized by variegated pigmentation, variable immune deficiencies, and platelet dysfunction. There are 8 known variants of HPS in humans. Lysosomes are membrane-bound organelles that contain hydrolytic enzymes necessary for intracellular degradation and represent the main cellular degradation compartment within the vacuolar system of the eukaryotic cell 1.Biogenesis of lysosomes partly depends on the function of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), −2, and −3 proteins2. The mutated proteins known to give rise to the HPS variants occur in one or more of the cytosolic BLOC complexes, each being composed of different proteins, including the Adaptor Proteins 1–4 (AP1-4) 3. Also, since mutations in the AP3B1 gene encoding the b subunit of the AP3 complex cause the only HPS subtype which is associated with an immune disorder along with other lysosomal abnormalities 4, this indicates that AP3 is important for lysosome function within the immune system. In the absence of AP3, cytotoxic T lymphocyte (CTL) mediated killing is severely impaired and cytotoxic lysosomal granules are unable to move along microtubules when the CTL recognizes target cells 5.We report a case of Hermansky Pudlak Syndrome Type 2 in a consanguineous female infant of Arabic origin who presented with severe sepsis, occulocutaneous albinism, platelet nucleotide deficiency, neutropaenia and a homozygous balanced inversion of chromosome 5; inv(5)(p15q13). Further molecular genetic analysis revealed a breakpoint in the AP3B1 gene, thereby confirming a genetic diagnosis of HPS2. Case Report:A five month old female infant presented with respiratory sepsis requiring ventilation. Clinical examination revealed occulocutaneous albinism (Figure 1) and hepatosplenomegaly. Laboratory data revealed severe neutropenia (0.2×109/l), CD4 lymphopaenia (446/microlitre), normal immunoglobulins, marked reduction of platelet nucleotides and abnormal platelet aggregation studies. [Display omitted] There was no evidence of haemophagocytic syndrome at presentation. Neutrophil activity was normal and there was no evidence of B-cell dysfunction. Unexpectedly, her CTLs demonstrated normal lysosomal granule release on activation.Karyotyping showed pericentric balanced inversion in chromosome 5, inv (5)(p15q13). Parental karyotyping revealed heterozygous inversion of chromosome 5 at the same breakpoint. Array comparative genomic hybridization confirmed absence of copy number gain or loss in the inversion. Bacterial artificial chromosome cloning using Fluorescent in situ hybridization confirmed the breakpoint to be within the APB3B1 gene.She made a good recovery from her initial sepsis and continues to remain neutropaenic and CD4 lymphopaenic on follow up for 12 months, and has required a number of admissions with viral sepsis (adenovirus and respiratory syncitial virus).In summary, this is a first report of homozygous pericentric balanced inversion of chromosome 5 inv(5)(p15q13), leading to disruption of the AP3B1 gene, giving rise to a clinical phenotype of Hermansky Pudlak Syndrome Type 2. The normal cytotoxic lymphocyte function remains unexplained. Further molecular genetic work is in progress to map the breakpoint in the AP3B1 gene and the corresponding gene(s) involved in the inversion. Disclosures:No relevant conflicts of interest to declare.

Cellular mechanisms underlying LPS-induced bactericidal activity in Dictyostelium discoideum (162.15)

Abstract Cells of the innate immune system detect pathogen-associated molecular patterns (PAMPs) through highly conserved pattern-recognition machinery. The conservation of this machinery allows for its characterization in model organisms. The unique model organism Dictyostelium discoideum can exist as single-celled amoebae that phagocytize bacteria for nutritional uptake. However, it has not been determined if D. discoideum uses pattern-recognition machinery to detect PAMPs. Here we show that D. discoideum mounts a response against the bacterial cell wall PAMP, lipopolysaccharide (LPS). LPS treatment results in an increased clearance of phagoctyzed bacteria by D. discoideum. LPS-induced bactericidal activity appears to be dependent on MAPK and Tir domain-containing protein A (TirA)-mediated pathways. Recent results suggest that increased bactericidal activity upon treatment with LPS may involve the induction and maturation of autophagosomes. We show here, by following the delivery of the autophagosomal marker Atg8 to degradative compartments, that Staphylococcus aureus induces autophagosomal maturation in D. discoideum only upon addition of LPS. We are currently characterizing the signaling pathways underlying bacterial induction of autophagy in D. discoideum. Characterization of the cellular events induced upon microbial pattern recognition in D. discoideum should provide valuable insight into conversed cellular mechanisms underlying microbial detection and killing.

Bacillus sphaericus Binary Toxin Elicits Host Cell Autophagy as a Response to Intoxication

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

Facilitation of μ-Opioid Receptor Activity by Preventing δ-Opioid Receptor-Mediated Codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane isa potential strategy to improve opioid analgesic therapies.

Differential Effects of Human Immunodeficiency Virus Type 1 Vpu on the Stability of BST-2/Tetherin

BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.

Vesicle degradation in dendrites of magnocellular neurones of the rat supraoptic nucleus

The magnocellular neurones of the supraoptic nucleus (SON) and paraventricular nucleus release neuropeptide from their axon terminals and also from their dendrites. In the axon terminals, swellings known as Herring bodies are responsible for the degradation of aged, unreleased large dense-cored vesicles (LDCVs) by lysosomes. Dendrites of magnocellular neurones also contain a large number of LDCVs but specialised areas of vesicle degradation have yet to be discovered. Using immunofluorescence labelling for lysosomes in vasopressin-enhanced green fluorescent protein (vasopressin-eGFP) transgenic rats, we found that lysosomes are preferentially located in the centre of the dendrites where there was a high density of vasopressin-eGFP expression. These data suggest that there are local “hot spots”, but not specific compartments for vesicle degradation in magnocellular dendrites.

Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2*

The human low affinity FcγRII family includes both the activating receptor FcγRIIA and the inhibitory receptor FcγRIIB2. These receptors have opposing signaling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. We demonstrate that upon engagement by multivalent aggregated human IgG, FcγRIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while FcγRIIB2 dissociates from the ligand and is routed separately into the recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase activity or ubiquitylation of receptor lysine residues. The sorting of FcγRIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of FcγRIIA and FcγRIIB2, the receptors are sorted independently to distinct final fates after dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behavior of different Fcγ receptors.

Dorsal Ruffle Microdomains Potentiate Met Receptor Tyrosine Kinase Signaling and Down-regulation

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4-acetamido-4'-isothiocyabatostilbene-2',2-disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation.

Coxiella burnetii Phase I and II Variants Replicate with Similar Kinetics in Degradative Phagolysosome-Like Compartments of Human Macrophages

Coxiella burnetii infects mononuclear phagocytes, where it directs biogenesis of a vacuolar niche termed the parasitophorous vacuole (PV). Owing to its lumenal pH (approximately 5) and fusion with endolysosomal vesicles, the PV is considered phagolysosome-like. However, the degradative properties of the mature PV are unknown, and there are conflicting reports on the maturation state and growth permissiveness of PV harboring virulent phase I or avirulent phase II C. burnetii variants in human mononuclear phagocytes. Here, we employed infection of primary human monocyte-derived macrophages (HMDMs) and THP-1 cells as host cells to directly compare the PV maturation kinetics and pathogen growth in cells infected with the Nine Mile phase I variant (NMI) or phase II variant (NMII) of C. burnetii. In both cell types, phase variants replicated with similar kinetics, achieving roughly 2 to 3 log units of growth before they reached stationary phase. HMDMs infected by either phase variant secreted similar amounts of the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha. In infected THP-1 cells, equal percentages of NMI and NMII PVs decorate with the early endosomal marker Rab5, the late endosomal/lysosomal markers Rab7 and CD63, and the lysosomal marker cathepsin D at early (8 h) and late (72 h) time points postinfection (p.i.). Mature PVs (2 to 4 days p.i.) harboring NMI or NMII contained proteolytically active cathepsins and quickly degraded Escherichia coli. These data suggest that C. burnetii does not actively inhibit phagolysosome function as a survival mechanism. Instead, NMI and NMII resist degradation to replicate in indistinguishable digestive PVs that fully mature through the endolysosomal pathway.

A dual function of V0-ATPase a1 provides an endolysosomal degradation mechanism in Drosophila melanogaster photoreceptors

The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6

Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.