γ-Secretase catalyzes the final cleavage of the β-amyloid precursor protein to generate amyloid-β peptide, the principal component of amyloid plaques in the brains of patients suffering from Alzheimer’s disease. Here, we review the identification of γ-secretase as a protease complex and its assembly and trafficking to its site(s) of cellular function. In reconstitution experiments, γ-secretase was found to be composed of four integral membrane proteins, presenilin (PS), nicastrin (NCT), PEN-2 and APH-1 that are essential and sufficient for γ-secretase activity. PS, which serves as a catalytic subunit of γ-secretase, was identified as a prototypic member of novel aspartyl proteases of the GxGD type. In human cells, γ-secretase could be further defined as a heterogeneous activity consisting of distinct complexes that are composed of PS1 or PS2 and APH-1a or APH-1b homologues together with NCT and PEN-2. Using green fluorescent protein as a reporter we localized PS and γ-secretase activity at the plasma membrane and endosomes. Investigation of γ-secretase complex assembly in knockdown and knockout cells of the individual subunits allowed us to develop a model of complex assembly in which NCT and APH-1 first stabilize PS before PEN-2 assembles as the last component. Furthermore, we could map domains in PS and PEN-2 that govern assembly and trafficking of the complex. Finally, Rer1 was identified as a PEN-2-binding protein that serves a role as an auxiliary factor for γ-secretase complex assembly.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.

γ‐Secretase is involved in the production of amyloid β‐peptide, which is the principal component of amyloid plaques in the brains of patients with Alzheimer disease. γ‐Secretase is a complex composed of presenilin (PS), nicastrin, anterior pharynx‐defective phenotype 1 (Aph1) and PS enhancer 2 (Pen2). We previously proposed a mechanism of complex assembly by which unassembled subunits are retained in the endoplasmic reticulum (ER) and only the fully assembled complex is exported from the ER. We have now identified Retention in endoplasmic reticulum 1 (Rer1) as a protein that is involved in the retention/retrieval of unassembled Pen2 to the ER. Direct binding of unassembled Pen2 to Rer1 is mediated by the first transmembrane domain of Pen2, and a conserved asparagine in this domain is required. Downregulation of Rer1 leads to increased surface localization of Pen2, whereas overexpression of Rer1 stabilizes unassembled Pen2. To our knowledge, Rer1 is the first identified interaction partner of mammalian transmembrane‐based retention/retrieval signals.

The Notch signaling pathway is an important regulation system for the development and self-renewal of different tissues. A specific feature of this signaling cascade is the function of Notch as a surface receptor and regulator of gene expression. Hence, Notch activation and signal transduction requires the proteolytic release of the Notch intracellular domain (NICD), which activates the transcription of cell-specific genes after its transport into the nucleus. To date, little is known about the mechanisms that mediate NICD nuclear import. We here show that transport of NICD into the nucleus is mediated by the canonical importin α/β1 pathway. GST pull-down experiments revealed that NICD binds via one of its four potential nuclear localization signals to importins α3, α4, and α7, but not to α1 and α5. siRNA-mediated knockdown experiments showed that importins α3, α4 (and to a lesser extent, α7) mediate nuclear import of NICD and thus are directly involved in Notch signaling.

Atlastins (ATLs) are membrane-bound GTPases involved in shaping of the endoplasmic reticulum (ER). Mutations in ATL1 and ATL3 cause spastic paraplegia and hereditary sensory neuropathy. We here show that the sensory neuropathy causing ATL3 Y192C mutation reduces the complexity of the tubular ER-network. ATL3 Y192C delays ER-export by reducing the number of ER exit sites, reduces autophagy, fragments the Golgi and causes malformation of the nucleus. In cultured primary neurons, ATL3 Y192C does not localize to the growing axon, resulting in axon growth deficits. Patient-derived fibroblasts possess a tubular ER with reduced complexity and have a reduced number of autophagosomes. The data suggest that the disease-causing ATL3 Y192C mutation affects multiple ER-related pathways, possibly as a consequence of the distorted ER morphology.