The growth-associated protein-43/B-50 (B-50/GAP-43) is conveyed from the neuronal soma into the axon by fast axonal transport and moved to the nerve terminal. To visualize and determine the type of vesicles by which B-50/GAP-43 is anterogradely transported in the regenerating rat sciatic nerve, we have investigated Lowicryl HM20 embedded nerve pieces dissected from the proximal side of a collection ligature. Ultrastructurally, numerous vesicular profiles of various sizes, tubules and mitochondria were seen to accumulate proximal to the collection ligature. Both, in unmyelinated and myelinated axons, B-50/GAP-43 immunoreactivity was associated with vesicular profiles which had a diameter of 50 nm. A fraction of the B-50/GAP-43 label co-localized with the small vesicle marker synaptophysin. Co-localization of B-50/GAP-43 was not detected with the large dense-core vesicle marker calcitonin gene-related peptide. These results indicate that, in rat sciatic nerve axons, B-50/GAP-43 is anterogradely transported in small 50 nm vesicles of the constitutive pathway. These transport vesicles were distinguished in two types. We suggest that one type carrying, both, B-50 GAP-43 and synaptophysin has as destination the nerve terminal, whereas the second type, which only contains B-50/GAP-43 and no synaptophysin, may be primarily targeted to the axolemma for local membrane fusion.
γ-Secretase is a promiscuous aspartyl protease responsible for the final intramembrane cleavage of various type I transmembrane proteins after their large ectodomains are shed. The vast functional diversity of its substrates, which are involved in cell fate decisions, adhesion, neurite outgrowth and synapse formation, highlights the important role γ-secretase plays in development, neurogenesis and neurodegeneration. The most renowned substrates are the amyloid precursor protein and Notch, from which γ-secretase liberates amyloid β peptides and induces downstream signaling, respectively. γ-Secretase is a multiprotein complex containing presenilin -which harbours the catalytic site-, nicastrin, APH-1 and PEN-2. Despite the efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of γ-secretase complexes. Given the multisubunit character of γ-secretase, we hypothesize that assembly must be regulated by additional factors early in the biosynthetic compartments. γ-Secretase complex assembly was studied in HeLa cells and wild-type or nicastrin-/-, presenilin-/- and APH-1-/- mouse embryonic fibroblasts (MEF) using a variety of molecular biological approaches including site-directed mutagenesis, siRNA, ectopic overexpression and protein electrophoresis. Our recent findings indeed support the working hypothesis that assembly of this complex occurs during ER-Golgi recycling and is at least mediated through interactions of nicastrin with the cargo retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 significantly increased binding of nicastrin to Rer1p demonstrating the competitive nature of these interactions. Moreover, downregulation of Rer1p promoted while overexpression decreased the formation of full complexes as assessed uby blue-native electrophoresis. Our data indicate that Rer1p expression levels control the formation of γ-secretase (sub)complexes (and concomitantly total cellular γ-secretase activity) at a very early stage of complex assembly namely in the formation of the nicastrin-APH-1 subcomplex. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly during active ER-Golgi recycling. This indicates that total cellular γ-secretase activity is restrained by a secondary ER quality control system that provides a potential therapeutic value.
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 β1,4-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).
We describe two patients with a cerebrocostomandibular-like syndrome and a novel mutation in conserved oligomeric Golgi (COG) subunit 1, one of the subunits of the conserved oligomeric Golgi complex. This hetero-octameric protein complex is involved in retrograde vesicular trafficking and glycosylation. We identified in both patients an intronic mutation, c.1070+5G>A, that disrupts a splice donor site and leads to skipping of exon 6, a frameshift and a premature stopcodon in exon 7. Real-time reverse transcriptase polymerase chain reaction showed in the first patient only 3% of normal transcript when compared with control. A delay in retrograde trafficking could be demonstrated by Brefeldin A treatment of this patient's fibroblasts. The costovertebral dysplasia of the two patients has been described in cerebrocostomandibular syndrome (CCMS), but also in cerebrofaciothoracic dysplasia and spondylocostal dysostosis. CCMS itself is heterogeneous because both autosomal dominant and autosomal recessive inheritance has been described. We anticipate further genetic heterogeneity because no mutations in COG1 were found in two additional patients with a CCMS.
Processing of the amyloid precursor protein (APP) leads to the production of amyloid‐β (Aβ), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin‐1 (PS‐1) plays a key role in the final step of Aβ formation, the γ‐secretase cleavage. Previously, we showed that PS‐1 is retained in pre‐Golgi compartments by incorporation into COPI‐coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS‐1 also mediates the retention of the β‐cleavage‐derived APP‐C‐terminal fragment (CTFβ) and/or Aβ in pre‐Golgi membranes. Overexpression of PS‐1 increased the percentage of CTFβ and/or Aβ in VTCs as well as their distribution to COPI‐coated VTC membranes. By contrast, overexpression of the dominant‐negative aspartate mutant PS‐1 D257A or PS‐knockout decreased incorporation of these APP derivatives into COPI‐coated membranes. Sorting of APP derivatives to COPI‐coated VTC membranes was not depending on the APP cytosolic tail. In post‐Golgi compartments, PS‐1 expression enhanced the association of full‐length APP/APPs with endosomal compartments at the expense of plasma membrane‐bound APP. We conclude that PS‐1, in addition to its role in γ‐secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS‐1 in this role may have important consequences for the progress of AD.
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. 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.
