Early Biosynthetic Compartments Research Articles

SEC24 isoform specificity regulates the assembly of γ‐secretase from dimeric subcomplexes

AbstractBackgroundγ‐Secretase is a diaspartyl protease with presenilin (PSEN) as the catalytic subunit. It targets a plethora of type I transmembrane proteins and, as such, affects a broad range of physiological processes. Because of this, aberrant functioning of γ‐secretase is linked to several diseases including cancer and Alzheimer’s disease (AD). Herein, mutations in the PSEN genes are associated with rare cases of familial AD and shift the production to longer, more aggregation prone amyloid β peptides. γ‐Secretase consists, besides the catalytic component PSEN, of nicastrin, APH1 and PEN‐2 that are co‐translationally translocated in the endoplasmic reticulum (ER). As γ‐secretase has a stoichiometry of 1:1:1:1, the assembly process is spatially and temporally regulated. Although it is generally agreed that this occurs in early biosynthetic compartments, it remains far from understood whether full assembly occurs already in the ER and to what extent Golgi‐to‐ER recycling is involved.MethodWe used advanced cell fractionation protocols and an in vitro reconstituted ER budding assay to explore in detail the stepwise assembly process of γ‐secretase in early biosynthetic compartments.ResultCell fractionation combined with blue native electrophoresis shows that subcomplexes are mainly present in ER‐enriched fractions while full complexes appear in the ER‐Golgi intermediate compartment, indicating that final assembly occurs beyond the ER. In vitro reconstitution of ER export reveals that none of the γ‐secretase subunits is required for ER‐exit of the others. However, knock‐out of any subunit induces the accumulation of preceding subcomplexes in de novo formed COPII vesicles. Mutating a motif in the cytosolic loop domain of PSEN1, which is lost in the familial AD‐associated PSEN1∆E9 mutant, abrogates ER‐exit of PSEN1 as well as PEN‐2, but not of nicastrin. This is explained molecularly by the selectivity of Sec24A/B and Sec24C/D in packaging PSEN1 and nicastrin, respectively, and further argues against full complex assembly prior to ER‐exit.ConclusionIn conclusion, our data support a model wherein individual subunits and dimeric subcomplexes preferentially exit the ER. Full assembly likely occurs in intermediate compartment/cis‐Golgi allowing complexes to escape active Golgi‐to‐ER recycling as part of a secondary quality control mechanism in controlling cellular γ‐secretase levels and activity.

Protein quality control by Rer1p in the early secretory pathway: from mechanism to implication in Alzheimer’s disease

γ-Secretase-mediated production of amyloid β from the amyloid precursor protein is recognized as a central player in the neuropathogenesis of Alzheimer’s disease (AD). One of the most peculiar features of this enzymatic activity is the fact that it targets transmembrane domains of mostly type I integral membrane proteins and thus manages to proteolyse peptide bonds within the hydrophobic lipid bilayers. In addition, γ-secretase does not exert its activity solely towards amyloid precursor protein, but to an increasing number of membrane proteins, including Notch, cadherins, syndecans, and so on. Because of the requirement of intramembrane proteolysis for a plethora of signaling pathways and cellular processes during embryonic development and organ physiology, this enzyme has drawn a lot of attention in the past 20 years. γ-Secretase is a multimeric transmembrane complex consisting of the catalytic presenilin, nicastrin, presenilin enhancer 2 (PEN2) and anterior-pharynx defective-1 (APH1) subunits. Proper assembly into functional complexes requires quality control mechanisms associated with the early biosynthetic compartments and allows mature complexes to transit to distal compartments where its activity is required. We previously identified Retrieval to ER protein 1 (Rer1p) as the first negative regulator of the stepwise assembly of γ-secretase during endoplasmic reticulum-to-Golgi transport. We review here the state of the art on how Rer1p regulates complex assembly, particularly γ-secretase, and evaluate the therapeutic potential of such regulatory processes in the context of AD.

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.

Presenilin‐1‐mediated Retention of APP Derivatives in Early Biosynthetic Compartments

Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), 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 Abeta formation, the gamma-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 beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta 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 gamma-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.

Presenilin 1 Is Required for Maturation and Cell Surface Accumulation of Nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.

Processing of β-Secretase by Furin and Other Members of the Proprotein Convertase Family

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.

Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to β-strands and α-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to beta-strands and alpha-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

Characterization of two arylsulfatase A missense mutations D335V and T274M causing late infantile metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. We describe a novel missense mutation in exon 6 causing the substitution of Asp335 by Val. In transient transfections no enzyme activity could be expressed from the arylsulfatase A cDNA carrying this mutation. Examination of the effects of the mutation in cells stably overexpressing the mutant enzyme revealed, that the mutant enzyme is catalytically inactive and degraded in an early biosynthetic compartment. We have also investigated the effects of a previously identified mutation (T274M). The specific catalytic activity of the Met274 substituted arylsulfatase is reduced to about 35% of the normal enzyme when measured with an artificial substrate. Most of this enzyme is also degraded in an early biosynthetic compartment.

A missense mutation P136L in the arylsulfatase A gene causes instability and loss of activity of the mutant enzyme

Metachromatic leukodystrophy is a lysosomal storage disease caused by deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of an Ashkenazi Jewish patient suffering from the severe late infantile form of the disease revealed a point mutation in exon 2 causing proline 136 to be substituted by leucine. The patient was homozygous for this mutation. Studies on Ltk- cells stably expressing the mutant enzyme show that the mutation causes complete loss of enzyme activity and rapid degradation in an early biosynthetic compartment.

Human Resident Langerhans Cells Display a Lysosomal compartment Enriched in MHC Class II

Langerhans cells are the antigen-presenting cells of the skin, belonging to the family of dendritic cells, which present exogenous antigens in the context of major histocompatibility complex class II (MHC-II) molecules to CD4+ T lymphocytes. Langerhans cells are potent stimulators of different T-cell responses including primary immune responses. Culturing of Langerhans cells leads to modulation of their phenotype and function, as they seem more capable of activating T cells, whereas freshly isolated Langerhans cells are specialized in the endocytosing and processing of antigen. We studied the intracellular distribution of MHC-II molecules and invariant chain (I-chain) in resident Langerhans cells using immunogold labeling of ultrathin cryosections of human epidermis and found the majority of intracellular MHC-II molecules present on membranes of rough endoplasmic reticulum and in so-called MHC-II-enriched compartments (MIIC). The MIIC appeared to be negative for the cation-independent mannose 6-phosphate receptor and positive for the lysosomal enzyme beta-hexosaminidase and acquired the endocytotic tracer, cationized horseradish peroxidase, only after 60 min of internalization. Taken together, these data show that MIIC in Langerhans cells share characteristics with lysosomes. I-chain, which is associated with MHC-II molecules in early biosynthetic compartments, was found in the rough endoplasmic reticulum and Golgi complex, but was detected only occasionally in MIIC and at the plasma membrane. MIIC with internal membrane vesicles showed some I-chain labeling, suggesting that these are newly formed MIIC in which degradation of the I-chain is not yet complete.

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption

Ligand binding activity of intrinsic factor-cobalamin receptor (IFCR) was determined in homogenates and isolated brush-border membranes (BBM) of ileum and kidney from dogs exhibiting simple autosomal recessive inheritance of selective cobalamin malabsorption (Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991) Pediatr. Res. 29, 24-31). IFCR activity of affected dog ileal homogenates was 3-4-fold higher than normal whereas IFCR activity in affected dog kidney homogenates was one-tenth of normal. The recovery of IFCR activity in the BBM of ileum and renal cortex of affected dogs was 30- and 20-fold less than normal, respectively. The dissociation constant (Kd) for intrinsic factor-cobalamin was similar in BBM of both tissues and was the same in affected and normal dogs. In the affected dog ileal BBM, activities of alkaline phosphatase and sucrase-isomaltase and vesicular transport of glucose and Na(+)-taurocholate were normal. Immunoblots showed no IFCR cross-reactive material in the ileal or renal BBM of affected dogs. IFCR purified by affinity chromatography from kidney of both normal and affected dogs had an Mr = 230,000. However, amino acid analysis revealed that the affected dog IFCR had more lysine than the normal, and protease cleavage of the purified IFCRs revealed different peptide maps. Asparagine-linked oligosaccharides of both proteins were sensitive to peptide N-glycosidase F cleavage, but only the affected dog IFCR was endoglycosidase H sensitive. These results suggest that cobalamin malabsorption in this canine family is caused by inefficient BBM expression of IFCR due to a mutation of IFCR and its retention in an early biosynthetic compartment.

A Frameshift Mutation in a Patient with Tay-Sachs Disease Causes Premature Termination and Defective Intracellular Transport of the α-Subunit of β-Hexosaminidase

Mutations of the gene encoding the α-subunit of the lysosomal enzyme, β-hexosaminidase, are the cause of Tay-Sachs disease. We previously showed that fibroblasts from one patient (WG1051) synthesized an unstable α-subunit that was smaller than normal and appeared to be trapped in an early biosynthetic compartment (Zokaeem, G., Bayleran, J., Kaplan, P., Hechtman, P., and Neufeld, E. F. (1987) Am. J. Hum. Genet. 40, 537–547). We now have identified the mutation as a deletion of cytosine at position 1510 of the coding sequence. We first determined that the structural abnormality was at the carboxyl terminus of the protein and then sequenced the corresponding regions of the cDNA and genomic DNA after amplification by the polymerase chain reaction. The frameshift mutation, which is present on both alleles, causes premature termination four codons downstream, and the loss of a very hydrophilic stretch of 22 amino acids. Expression of α-subunit cDNA with the cytosine deletion in Cos-1 cells reproduced the WG1051 phenotype, i.e. a truncated α-subunit that was retained and degraded in an early compartment, presumably the endoplasmic reticulum. Loss of the cysteine residue at position 522 was not the sole cause of instability and defective transport.