Abstract
Trafficking of integral membrane proteins between the ER and Golgi complex, and protein sorting and trafficking between the TGN and endosomal/lysosomal compartments or plasma membranes, are dependent on cis-acting, linear amino acid sorting signals. Numerous sorting signals of this type have been identified in the cytoplasmic domains of membrane proteins, several of which rely on basic residues. A novel Golgi export signal that relies on a membrane-proximal polybasic motif (PBM) was recently identified in the reptilian reovirus p14 protein, a representative of an unusual group of bitopic fusion-associated small transmembrane (FAST) proteins encoded by fusogenic orthoreoviruses and responsible for cell-cell fusion and syncytium formation. Using immunofluorescence microscopy, cell surface immunofluorescence, and endoglycosidase H assays, we now show the p14 PBM can mediate several distinct trafficking functions depending on its proximity to the transmembrane domain (TMD). When present within 4-residues of the TMD it serves as a Golgi export signal, but when located at the C-terminus of the 68-residue p14 cytoplasmic endodomain it functions as an ER retention signal. The PBM has no effect on protein trafficking when located at an internal position in the cytoplasmic domain. When present in both membrane-proximal and -distal locations, the PBMs promote export to, and efficient retrieval from, the Golgi complex. Interestingly, the conflicting trafficking signals provided by two PBMs induces extensive ER tubulation and segregation of ER components. These studies highlight how a single trafficking signal in a simple transmembrane protein can have remarkably diverse, position-dependent effects on protein trafficking and ER morphogenesis.