Dictyostelium IQGAP-related Protein Specifically Involved in the Completion of Cytokinesis

The gapA gene encoding a novel RasGTPase-activating protein (RasGAP)–related protein was found to be disrupted in a cytokinesis mutant of Dictyostelium that grows as giant and multinucleate cells in a dish culture. The predicted sequence of the GAPA protein showed considerable homology to those of Gap1/Sar1 from fission yeast and the COOH-terminal half of mammalian IQGAPs, the similarity extending beyond the RasGAP-related domain. In suspension culture, gapA⁻ cells showed normal growth in terms of the increase in cell mass, but cytokinesis inefficiently occurred to produce spherical giant cells. Time-lapse recording of the dynamics of cell division in a dish culture revealed that, in the case of gapA⁻ cells, cytokinesis was very frequently reversed at the step in which the midbody connecting the daughter cells should be severed. Earlier steps of cytokinesis in the gapA⁻ cells seemed to be normal, since myosin II was accumulated at the cleavage furrow. Upon starvation, gapA⁻ cells developed and formed fruiting bodies with viable spores, like the wild-type cells. These results indicate that the GAPA protein is specifically involved in the completion of cytokinesis. Recently, it was reported that IQGAPs are putative effectors for Rac and CDC42, members of the Rho family of GTPases, and participate in reorganization of the actin cytoskeleton. Thus, it is possible that Dictyostelium GAPA participates in the severing of the midbody by regulating the actin cytoskeleton through an interaction with a member of small GTPases.     

FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis

Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G₁, when FOP protein levels are lowest, and most labeled in G₂. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.     

Furin Is Involved in Baculovirus Envelope Fusion Protein Activation

The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30-41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1 and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominantly as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.     

EtpB Is a Pore-Forming Outer Membrane Protein Showing TpsB Protein Features Involved in the Two-Partner Secretion System

Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane β-barrel domain that is occluded by an N-terminal α-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a β-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.     

SurA Is Involved in the Targeting to the Outer Membrane of a Tat Signal Sequence-Anchored Protein

The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii, the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of β-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli, periplasmic chaperones SurA, Skp, and DegP work together with the β-barrel assembly machinery (Bam) to target and insert β-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system.     

Translocator Protein 2 Is Involved in Cholesterol Redistribution during Erythropoiesis

Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis.Translocator protein (TSPO)² is an 18-kDa protein that was previously known as PBR (peripheral type benzodiazepine receptor) and represents a gene family evolutionarily conserved from bacteria to humans (1). In bacteria, TSPO is the tryptophan-rich sensory protein, an integral membrane protein that acts as a negative regulator of the expression of specific photosynthesis genes in response to oxygen and light (2). It is involved in the efflux of porphyrin intermediates from the cell, and several conserved aromatic residues within TSPO are thought to be involved in binding porphyrin intermediates (2). TSPO of bacterial origin has been shown to have the same ligand binding properties as mammalian TSPO proteins (3). In addition to the binding of porphyrin and heme, mammalian TSPO can replace the activity of its bacterial homologs (2, 4, 5). Rat TSPO was shown to retain its structure within the bacterial outer membrane, to functionally substitute for the bacterial homolog, and to act in a manner similar to TSPO in the outer mitochondrial membrane (6). Therefore, it is conceivable that some conserved functions of the Tspo genes within a cell are maintained from bacteria to plants and to mammals.In mammals, the biological significance of TSPO has been studied for decades, and TSPO has been shown to be involved in a variety of cellular functions, including cholesterol transport and steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and proliferation (7–10). Moreover, its expression correlates with certain pathological conditions such as cancer and endocrine and neurological diseases (8). Although some conserved cellular functions of Tspo are shared from bacteria to mammals, such as cholesterol-binding and transport, their biological significance seems to have adapted to serve specific functions critical for each organism. For instance, cholesterol transport into mitochondria is the rate-determining step in steroidogenesis (8, 11). TSPO serves the similar function in plants (12), insects (13), and mammals (14). However, the appearance of the drug, such as the benzodiazepine diazepam, binding sites on TSPO evolved later than the brain-specific γ-aminobutyric acid A receptor benzodiazepine binding sites (15), although drug binding was observed in both the plant and insect TSPOs (12, 13). Thus, throughout evolution, mammalian Tspo genes have exhibited extraordinary plasticity, a valuable trait to be further exploited.We sought to reveal the mechanisms controlling the molecular evolution of Tspo and Tspo-like genes and the ligand binding sites in recently sequenced mammalian and other eukaryotic genomes and characterize the relationships and potential functional similarities in cholesterol synthesis, trafficking, and cholesterol-supported steroidogenesis between different Tspo genes. During these studies, we identified a new family of Tspo-like genes involved in cholesterol trafficking and redistribution, which is linked to erythropoiesis and probably to a new mechanism of erythroblast maturation.     

A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ~160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ~1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.     

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca²⁺-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.     

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream-located pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered.     

The Conserved Yeast Protein Knr4 Involved in Cell Wall Integrity Is a Multi-domain Intrinsically Disordered Protein

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.     

The Cytosolic DnaJ-like Protein Djp1p Is Involved Specifically in Peroxisomal Protein Import

The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ. DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes. Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome. The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities. Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal. Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant. We conclude that Djp1p is specifically required for peroxisomal protein import.     

MODIFIED VACUOLE PHENOTYPE1 Is an Arabidopsis Myrosinase-Associated Protein Involved in Endomembrane Protein Trafficking

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-δ tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.The plant endomembrane system is a complex network of subcellular compartments that includes the endoplasmic reticulum (ER), Golgi apparatus, vacuole, plasma membrane, secretory vesicles, and numerous intermediary compartments. Protein trafficking through the endomembrane system requires specific cargo recognition and delivery mechanisms that are mediated by a series of highly specific targeting signals (Surpin and Raikhel, 2004), whose proper recognition is critical for the function of numerous downstream processes, such as floral development (Sohn et al., 2007), gravitropism (Kato et al., 2002; Surpin et al., 2003; Yano et al., 2003), abiotic stress tolerance (Zhu et al., 2002), autophagy (Surpin et al., 2003; Bassham., 2007), pathogen defense (Robatzek, 2007), and turgor pressure and growth (De, 2000).The importance of protein trafficking for plant survival was demonstrated by the identification of the essential Arabidopsis (Arabidopsis thaliana) gene VACUOLELESS1 (VCL1; Rojo et al., 2001). VCL1 was identified as a homolog of Saccharomyces cerevisiae VPS16, which is critical for yeast vacuole biogenesis. Knockouts of yeast VPS16 lack discernible vacuoles but survive despite their severe phenotype. The absence of vacuoles in Arabidopsis vcl1-1 mutants results in embryo lethality (Rojo et al., 2001). The essential nature of trafficking in plants was also demonstrated by insertional mutagenesis of syntaxin genes, where lethality was observed after disruption of single genes in families with highly homologous members (Lukowitz et al., 1996; Sanderfoot et al., 2001). Thus, despite large families of endomembrane components with many homologous genes, many are not redundant in Arabidopsis.Although embryo-lethal mutations provide critical data, it is difficult to obtain additional information. Less severe mutations have proven successful for functional genetics studies of endomembrane trafficking proteins. For example, point mutations in the KATAMARI1/MURUS3 (KAM1/MUR3; Tamura et al., 2005) and KATAMARI2/GRAVITROPISM DEFECTIVE2 (KAM2/GRV2; Tamura et al., 2007; Silady et al., 2008) genes lead to disruption of endomembranes, resulting in the formation of perinuclear aggregates containing organelles. Nonlethal trafficking disruptions have also been generated using chemical genomics, where small molecules were used to perturb trafficking of a soluble cargo protein (Zouhar et al., 2004) and localization of endomembrane markers (Surpin et al., 2005; Robert et al., 2008). Such studies have provided valuable clues about these essential cellular processes.In order to obtain less severe, viable mutants with defects in endomembrane protein trafficking, we previously identified point mutants with defects in localization of a tonoplast reporter protein, GFP:δ-TIP (Avila et al., 2003). Two hundred one putative mutants were grouped into four categories based on the nature of their defects. One unique mutant, cell shape phenotype1, was recently characterized as a trehalose-6-phosphate synthase with roles in regulation of plant architecture, epidermal pavement cell shape, and trichome branching (Chary et al., 2008).Here, we describe an endomembrane trafficking mutant categorized by perinuclear aggregates of GFP:δ-TIP fluorescence (Avila et al., 2003). We refer to this mutant as modified vacuole phenotype1-1 (mvp1-1). At least five endomembrane fusion proteins are partially relocalized to these structures. Positional cloning identified MVP1 as a myrosinase-associated protein (MyAP) localized previously to the tonoplast by proteomics (Carter et al., 2004). mvp1-1 mutants showed reduced endomembrane system functionality, as demonstrated by defects in growth, utilization of stored carbon, gravitropic responsiveness, salt sensitivity, and increased susceptibility to a fungal necrotroph. MVP1 interacted specifically with THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), a known myrosinase protein in Arabidopsis, and the mvp1-1 mutation had a significant effect on nitrile production during glucosinolate hydrolysis, suggesting a role in myrosinase function. Furthermore, MVP1 may function in quality control of glucosinolate hydrolysis by contributing to the proper tonoplast localization of TGG2.     

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.     

Force Generation in Lamellipodia Is a Probabilistic Process with Fast Growth and Retraction Events

Polymerization of actin filaments is the primary source of motility in lamellipodia and it is controlled by a variety of regulatory proteins. The underlying molecular mechanisms are only partially understood and a precise determination of dynamical properties of force generation is necessary. Using optical tweezers, we have measured with millisecond (ms) temporal resolution and picoNewton (pN) sensitivity the force-velocity (Fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. When force and velocity are averaged over 3-5 s, the Fv relationships can be flat. On a finer timescale, random occurrence of fast growth and subsecond retractions become predominant. The maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 μm is 10⁻¹⁶ W. Our results clarify the dynamical properties of force generation: i), force generation is a probabilistic process; ii), underlying biological events have a bandwidth up to at least 10 Hz; and iii), fast growth of lamellipodia leading edge alternates with local retractions.     

Macroautophagy Is Not Directly Involved in the Metabolism of Amyloid Precursor Protein

Alterations in the metabolism of amyloid precursor protein (APP) are believed to play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate that APP is proteolytically processed in endosomal-autophagic-lysosomal compartments. In this study, we used both in vivo and in vitro paradigms to determine whether alterations in macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse models; however, discrepancies between LC3-II and APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous murine Aβ40 from RIPA-soluble extracts was increased in brains of all three mice. The in vivo relationship between AV and APP-CTF accumulation was also seen in cultured neurons treated with agents that impair primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by agents that induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive lysosomes. These data suggest that neuronal macroautophagy does not directly regulate APP metabolism but highlights the important anti-amyloidogenic role of lysosomal proteolysis in post-secretase APP-CTF catabolism.