Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS. ALS1 is a fatal neuromuscular disease that presents as weakness, spasticity, and muscle atrophy. The disease is caused by selective degeneration of motor neurons in the brain, brainstem, and spinal cord. Although ALS presents mostly as a sporadic disease, a familial form of ALS is seen in ϳ10% of cases. Twenty percent of familial ALS (FALS) cases are caused by point mutations in the SOD1 gene. More than 90 distinct amino acid mutations spread throughout the sequence of this 153-residue protein have been identified (1). The finding that many FALS-associated SOD1 mutants possess full specific enzyme activity (2) suggests that the disease is not caused by loss of normal dismutase activity. Further support for this idea has come from transgenic mice studies. Transgenic mice that harbor FALS-associated SOD1 mutations develop ALS-like symptoms despite having greater than normal levels of SOD1 activity, including the normal complement of endogenous mouse SOD1 enzyme (3). Furthermore, SOD1 knockout mice do not develop ALS-like symptoms. Thus, it has been proposed that mutations in SOD1 cause FALS by a gain, rather than a loss, of function (reviewed in Ref.

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg2+ATP and Mg2+GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins α2p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of α2p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of α2p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPγS, inhibition by Brefeldin A (BFA), or depletion of β-COP from cytosol. Therefore, the p24 family member, α2p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

Abstract. The intrahepatic distribution of apolipoprotein E has been assessed by immunogold labeling of cryosections as well as by Western blotting of organelles isolated from liver homogenates. Both techniques supported the prior analytical fractionation studies of Wong (1989) who concluded that intrahepatic apoE was largely endosomal. All endosomal components decorated by gold particles indicative of apoE antigenicity in cryosections appeared filled with lipoprotein-like particles thereby accounting for this prominent morphological feature of isolated liver endosomes. The distribution of gold particles about the hepatic Golgi apparatus revealed a high content of apoE in closely apposed endosomes, ca. 400 nm in diameter, double labeled for apoE and internalized HRP. Remarkably, apoE (but not internalized HRP) was also observed within saccular distensions of all saccules of stacked Golgi cisternae but absent from the flattened saccular components as was also observed for apoB. This contrasted with albumin, the major secretory protein, which was uniformly distributed throughout the hepatic Golgi apparatus. These observations support a growing body of evidence for intra-Golgi sorting of secretory material in hepatic Golgi apparatus. The lack of any immunoreactive apoE or albumin in small 70-90 nm vesicles about the Golgi cisternae suggests limits to current models of vesicle-mediated intra-Golgi transport.

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged–GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the β-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

BackgroundThe Aβ peptide that accumulates in Alzheimer’s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by β- and γ-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent Aβ production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and Aβ production.ResultsIn this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into Aβ, while knockdown of LRP10 expression increases Aβ production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP β-cleavage into Aβ. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.ConclusionsThe present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer’s disease.