Two Motifs Target Batten Disease Protein CLN3 to Lysosomes in Transfected Nonneuronal and Neuronal Cells

Abstract

Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.

The transmembrane topology of Batten disease protein CLN3 determined by consensus computational prediction constrained by experimental data

“…We have shown that a consensus approach combined with careful analysis of evolutionary data can produce a model which agrees with almost all published experimental data. Previous work had been less confident about the number of possible TM helices, although one model proposed on the basis of experimental findings [13] agrees closely with ours. Our unexpected finding that orthologues of CLN3 might produce different topologies highlight the increasing realization that transmembrane topology may not be as highly conserved between distantly related species as previously thought.…”

“…This region contains two distinctive lysosomal sorting motifs [13] that experimentally must reside in the cytosol.…”

“…This bias in frequency towards lumenal loops may be explained by the ability of glycosylation to prevent proteolysis in the protease-rich lysosomal lumen, thought to be the reason lysosomal membrane proteins are often heavily glycosylated. Both CLN3 and its yeast orthologues traffic to the lysosome/vacuole [13,18], although this may not be their only functional location. The remaining potential N-glycosylation motif has been placed within the first TM helix; however, unlike the other three, this site has not been validated experimentally and is most likely a false positive.…”

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms

“…Therefore, we sought to demonstrate that PfCRT's C-terminus was exposed in the cytoplasm, which would be consistent with localization in the lysosomal membrane. Digitonin was used to selectively permeabilize the plasma membrane without disturbing the integrity of the lysosomal membrane [29]. We demonstrated selective plasma membrane permeabilization using tubulin as a cytoplasmic marker and cathepsin as an intralysosomal marker.…”

“…Cells were incubated for 1 hr at room temperature (RT) with slow agitation with primary antibodies, and for 30 min each with secondary and tertiary reagents, followed by 4 washes in PBS of 5 min each. Selective plasma membrane permeabilization was achieved using the method of Kyttala and coworkers [29]. After the final wash, PBS was replaced by mounting medium (Sigma, MO) and sealed under a plastic coverslip.…”