Decoding the trafficking motifs in the mammalian VNUT N- and C-Termini

Rationale: The vesicular nucleotide transporter (VNUT) is crucial for loading adenosine triphosphate (ATP) into vesicles. ATP acts as a neurotransmitter in the central and peripheral nervous systems and is implicated in diverse physiological functions. We have shown that mammalian VNUT localises to the Golgi apparatus and endoplasmic reticulum (ER), diverging from the vesicular localisation of other neurotransmitter transporters. Objective: To identify trafficking motifs in VNUT's termini that explain its unexpected subcellular localisation. Methods: Bioinformatics analyses mapped phylogenetic changes in VNUT's C-terminus and uncovered a putative N-terminal retrograde transport motif. We used chimeric and mutagenic analyses to dissect functional motifs in regions of the N- and C-termini of VNUT. Results: Epifluorescence microscopy revealed that mammalian C-terminal motifs promote ER-Golgi retention compared to avian (tyrosine, dileucine) motifs. Truncation of the putative N-terminal COPI-interacting motif disrupted retrograde Golgi-ER transport. Conclusions: Terminal motifs mediate mammalian VNUT trafficking, advancing our understanding of purinergic signalling.