Abstract
The cellular prion protein (PrPC) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrPC and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrPC. The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrPC.
Highlights
Prion disorders are invariably fatal diseases of humans and animals [1]
In search of physiological interactors of PrPC, we recently observed that two members of the family of ZIP zinc transporters [3], ZIP6 (Slc39a6) and ZIP10 (Slc39a10), co-purified with PrPC [4] and, subsequently, we discovered that the prion gene family descended in evolution from an ancestral ZIP gene [5]
To minimize the risk that off-target binding might confound data interpretation when comparing the localization of the three LIV-1 subfamily of ZIP zinc transporters (LZTs) with proteinspecific antibodies by confocal immunofluorescence microscopy, a C-terminal HA tag was used for detection after transient transfection in N2a cells (Figure 1A)
Summary
Prion disorders are invariably fatal diseases of humans and animals [1]. In prion diseases, the host-encoded cellular prion protein (PrPC) undergoes a conformational transition to a diseaseassociated ‘scrapie’ conformer, commonly referred to as PrPSc [2]. ZIP zinc transporters are encoded by the slc39a gene family that consists of fourteen genes in humans and mice. ZIP6 and ZIP10, together with their phylogenetically closest paralog ZIP5 (Slc39a5), are members of the LIV-1 subfamily of ZIP zinc transporters (LZTs) that are characterized by a conserved sequence motif found in transmembrane domain V [6] and the presence of N-terminal ectodomains absent in non-LZT members of this protein family. The mechanism of evolution of the PrP founder gene from an ancestral LZT gene was most likely based on a retrotransposition event that led to a loss of sequences coding for the C-terminal multi-spanning transmembrane domain present in LZTs [7]. Pair-wise alignments indicate that, in particular, LZT and PrP sequences found in fish genomes have retained considerable sequence similarity (up to 41%) [5]
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