Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.

The hetero-octameric conserved oligomeric Golgi (COG) complex is essential for the structure/function of the Golgi apparatus through regulation of membrane trafficking. Here, we describe a patient with a mild form of a congenital disorder of glycosylation type II (CDG-II), which is caused by a homozygous nonsense mutation in the hCOG8 gene. This leads to a premature stop codon resulting in a truncated Cog8 subunit lacking the 76 C-terminal amino acids. Mass spectrometric analysis of the N- and O-glycan structures identified a mild sialylation deficiency. We showed that the molecular basis of this defect in N- and O-glycosylation is caused by the disruption of the Cog1-Cog8 interaction due to truncation. As a result, Cog1 deficiency accompanies the Cog8 deficiency, preventing assembly of the intact, stable complex and resulting in the appearance of smaller subcomplexes. Moreover, levels of beta1,4-galactosytransferase were significantly reduced. The defects in O-glycosylation could be fully restored by transfecting the patient's fibroblasts with full-length Cog8. The Cog8 defect described here represents a novel type of CDG-II, which we propose to name as CDG-IIh or CDG caused by Cog8 deficiency (CDG-II/Cog8).

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for ␥-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membranebound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where ␥-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for ␥-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.Neurotrophins belong to a small family of neurotrophic factors that include nerve growth factor (NGF), 4 brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4). They regulate different aspects of the developing and adult nervous system by binding to specific members of the Trk family of receptor-tyrosine kinases (TrkA, -B, and -C) or to the p75 neurotrophin receptor (p75). Their involvement includes neuronal migration, cell death, axonal elongation, myelinization, neuronal differentiation, and synaptic plasticity (1-3). p75 is a multifunctional type I transmembrane protein that is structurally related to the tumor necrosis factor receptor superfamily. It binds all neurotrophins, alone or in complex with Trk receptors, but also other ligands such as amyloid peptides and pro-neurotrophins (4, 5). The association of p75 with different receptors shapes the outcome of a signaling event. For example, binding of p75 to TrkA in the presence of NGF promotes neuronal survival, whereas interaction with the Nogo receptor (NgR) in the presence of its ligands, such as Nogo and myelin-associated glycoprotein (MAG), results in growth cone collapse and the inhibition of axonal regeneration (1, 4, 5). In addition, different downstream p75-associated signaling cascades are triggered through the interaction of its cytosolic portion with multiple adaptor proteins (6). Neurotrophin binding to p75 also induces accumulation of p75 in recycling endosomes where it associates with specific p75 downstream signaling effectors (7-9). Hence trafficking of p75 in the cell body or in axons (10, 11) may determine downstream signaling of p75 as previously shown for Trk receptors (12). The regulation of p75 signaling becomes even more complicated with the finding that p75 is subject to a dual processing starting with shedding of the ectodomain and followed by ␥-secretase cleavage. Thi...

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni-and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway. Amyloid protein precursor (APP)1 is a type I membranespanning glycoprotein which is ubiquitously expressed in mammalian cells (1). Proteolytic processing of APP results in the generation of an ϳ40-residue amyloid (A␤) fragment which accumulates in the brains of individuals with Alzheimer's disease (AD). A central role for A␤ in the pathogenesis of AD is indicated by the discovery that various mutations within or flanking the A␤ region of APP cosegregate with affected status in individuals from several families with autosomal dominant AD. For this reason, the localization, trafficking, and processing of APP has generated great interest.Immunocytochemical studies of APP in cultured cells and in brain tissue reveal that a predominant fraction of APP is localized to the endoplasmic reticulum and the Golgi apparatus (2, 3). The localization of APP to biosynthetic organelles can be explained in part by the very high rate of synthesis and turnover of this protein. In axonal and synaptic compartments of brain tissue, APP is localized to large vesicular structures (2,3). APP is also found on the surface of cultured cells, from where it can be internalized and converted to A␤ (4 -7). APP is transported by fast axonal transport in central and peripheral neurons (8,9). Soluble APP (APP s ) can be released at synapses, 2 although it is not known whether the soluble fragments are generated at the synapse. Pools of unprocessed APP are also transported retrogradely from axonal or synaptic compartments to neuronal cell bodies and dendrites (10, 11). It is not known which membrane trafficking pathways are involved in anterograde and retrograde transport of APP in neurons or whether APP-containing vesicles are related to the recycling pathway of synaptic vesicles within the nerve terminal.In an attempt to study the nature of the APP-containing vesicles in nerve terminals, we have isolated APP-containing membranes from synaptosomes of rat forebrain and from PC12 cells. To differentiate vesicles participating in the synaptic vesicle pathway from other endocytic vesicles, organelles were immunoisolated using immobilized monoclonal antibodies directed against either synaptophysin, synaptobrevin, or the endosomal GTPase, Rab5. Synaptophysin and s...