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Traffic in Human Genetic Diseases

Sec23 has been implicated in a rare craniofacial disorder (CLSD) that causes various skeletal defects. The CLSD mutation maps to a residue on the surface of Sec23 that is away from contact with the membrane. Although the mutation, F382L, produces a conservative substitution, the effect of the allele on primary fibroblasts cultured from patients is profound, with a substantial distortion of the ER reminiscent of the original and lethal sec23-1 of yeast. Humans have two paralogs of Sec23, but cultured fibroblasts and calvarial osteoblasts, which may be responsible for the skeletal effects in CLSD patients, have only one copy, Sec23A. We have established a vesicle-budding reaction reconstituted with mammalian COPII proteins and ER membranes to explore CLSD and other unique features of human membrane protein traffic. Using pure recombinant proteins, we found that the CLSD mutation interferes with the binding and assembly of a scaffold complex, Sec13/31, which normally builds upon the inner coat of Sar1p and Sec23/24p.

Membrane proteins implicated in familial forms of Alzheimer's disease (FAD) are substrates for vesicular trafficking, and defects associated with protein transport may play a role in the pathology of AD. Presenilin 1 (PS1) is an essential subunit of an enzyme, gamma-secretase, that serves important roles in the maturation of proteins involved in signaling and development. However, mutant forms of PS1 cause unscheduled processing of amyloid precursor protein (APP) to generate an amyloidogenic peptide that accumulates in brain neuritic plaques that are characteristic of AD. Surprisingly, the PS1 mutations that potentiate the action of gamma-secretase on APP are spread throughout the molecule, including in domains exposed to the cytoplasm, to the lumen of the ER or extracellular space, and within the membrane bilayer. These alleles may produce forms of PS1 that fold improperly and retard the traffic of gamma-secretase from the ER. Such impaired traffic could influence the transport and proteolytic processing of APP.

In work initiated with support from private donors, including the Adler Foundation, we used mammalian COPII proteins to explore the packaging of APP and PS1 in lysates of fibroblast and neuronally derived cell lines. PS1 is enriched in the ER and Golgi membrane in precursor and autoproteolytically processed forms. Precursor forms of PS1 and the gamma-secretase complex are packaged along with APP into COPII vesicles, although the requirements for packaging are distinct. Cell lines derived from mouse PS-knockout strains are proficient in the packaging of APP. However, certain FAD mutant forms of PS1 retard the incorporation of mutant PS1 and apparently intact gamma-secretase complex. Retention of the gamma-secretase in the ER, or in a subsequent traffic organelle, could lead to aberrant processing of APP and to the production and secretion of the amyloidogenic peptide Abeta42. Our current work is focused on the role of the early endosome as a station for aberrant processing of APP.

Analysis of Vesicle Budding from the ER

Vesicle Traffic Late in the Secretory Pathway

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