The subcellular trafficking of the prion protein: Characterisation of the function of the PrPc N-terminus

Transmissible spongiform encephalopathies (TSEs) are degenerative
diseases of the central nervous system in humans and animals, and
include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep
and bovine spongiform encephalopathy in cattle. These spongiform
encephalopathies can manifest as sporadic, familial and acquired
disorders and are caused by the conformational alteration of the
non-pathogenic cellular prion protein (PrPc) into a infectious
isoform denoted PrPSc. The latter therefore represents a pathogenic
agent (prion) which does not contain nucleic acids. During
biogenesis, PrPc undergoes posttranslational modifications with the
addition of two N-linked carbohydrate chains and a
glycosyl-phosphatidyl-inositol-(GPI)-anchor. Properly folded PrPc
transits through the Golgi compartment and the secretory pathway
and is attached to the outer leaflet of the plasma membrane by the
GPI-anchor. The cellular function of the prion protein is still
unknown, although binding of copper to the octapeptide repeat
sequence located at its N-terminus suggests a role of PrPc related
to this phenomenon. In the present work, the physiological function
of the N-terminal part of PrPc in subcellular trafficking was
analysed. Metabolic labelling and surface-biotinylation assays were
performed in order to compare the intracellular trafficking and
turnover of PrPc mutants showing specific deletions within their
N-terminal sequence with those of wild type PrPc (wtPrPc). Upon
transient expression of these constructs in murine neuroblastoma
cells, these deletions, although not influencing the biochemical
properties or the cell surface expression of these proteins, lead
to a delay in their endocytosis. The prolongation of the
internalisation kinetics was shown to be dependent on the length of
the deletion: truncation of the complete N-terminus leads to the
almost complete inhibition of internalisation. The analysis of the
kinetics of degradation showed a similar correlation with the
N-terminal part of PrPc, since the half-life of the PrP-mutants was
significantly prolonged when compared to that of the wild type
protein. Additionally performed detailed analysis of the secretory
pathway with immunoprecipitation assays showed that N-terminally
truncated PrP molecules reach the plasma membrane at a later time
point, when compared with wtPrPc. A closer analysis of the
processing of the sugar molecules linked to these proteins
performing an Endo-H digestion revealed that this delay in the
transport to the cell surface takes place in a cellular compartment
following the mid-Golgi. The following studies were done with a
chimeric protein consisting of the short N-terminal segment of
Xenopus laevis, which does not contain the copper-binding
octarepeat region, fused to the N-terminally truncated mouse PrPc.
These studies showed that endocytosis of this protein and its
transport through the secretory pathway were comparable to those of
the mouse wtPrPc. It was therefore concluded that the N-terminus
belonging to a phylogenetically remote species can rescue the wild
type trafficking phenotype. These results indicate that the
N-proximal domain of the prion protein functions as a targeting
element and is essential for both transport to the plasma membrane
and modulation of endocytosis. The data support a model in which
the N-terminal part of PrPc represents an epitope for binding to a
transmembrane receptor containing internalisation-promoting motifs
or for inclusion of PrPc into the secretory raft-compartments. The
present work also indicates for the first time that copper affinity
of the octarepeats and subcellular trafficking represent separate
aspects in the life-cycle of the prion protein.