The Ins and Outs of Protein Trafficking Pathways: Insights from the FAST Proteins

Abstract

Sorting and trafficking of integral membrane proteins to the plasma membrane is essential for cellular homeostasis. Our understanding of the pathways and sorting signals that regulate protein trafficking is far from complete, particularly as it relates to protein exit from the Golgi. The reovirus fusion-associated small transmembrane (FAST) proteins are small integral membrane proteins that traffic through the ER-Golgi pathway to the plasma membrane where they cause cell-cell membrane fusion. The small sizes of FAST proteins and their simple structures provide an excellent system to identify factors and pathways affecting plasma membrane trafficking. Using the reptilian reovirus p14 FAST protein, I discovered that a polybasic motif (PBM), located four residues downstream of the transmembrane domain in the cytoplasmic tail of p14, is required for p14 export from the Golgi to the plasma membrane. Extensive mutagenesis of the PBM indicated that the number, but not the identity or position, of basic residues in the PBM directs p14 trafficking to the plasma membrane, with a minimum of three basic residues being required for efficient Golgi export. Insertion of the PBM into a Golgi-resident ERGIC-53 chimeric protein resulted in protein trafficking to the plasma membrane, indicating the p14 PBM functions as an autonomous Golgi export signal. I also discovered that the PBM can serve diverse trafficking roles depending on its proximity to the transmembrane domain. The PBM exerts no effect when located at internal positions in the 68-residue p14 cytoplasmic tail, it functions as an ER retention signal when located at the C-terminus, and when present at both membrane-proximal and -distal locations promotes export to and retrieval from the Golgi complex. Interestingly, the conflicting signals provided by membrane-proximal and -distal PBMs induce extensive ER tubulation and segregation of luminal ER components. A single trafficking motif can therefore exert remarkably diverse, position-dependent effects on protein trafficking and membrane compartment morphogenesis. To determine how the p14 PBM directs Golgi-plasma membrane transport, I examined the effects of various trafficking factors and pathways on this process. Yeast two-hybrid analysis identified Rab11A as a genetic interaction partner of p14. Co-immunoprecipitation (co-IP) determined that p14 interacts preferentially with GTP-bound activated Rab11A in a PBM-dependent manner. Overexpression of dominant-negative Rab11A, but not Rab5, significantly reduced p14 surface expression. Fluorescence resonance energy transfer (FRET) microscopy indicated activated Rab11 directly interacts with p14 dependent on the PBM, the first example of activated Rab11 directly interacting with membrane cargo for Golgi-plasma membrane trafficking. Furthermore, RNA interference revealed that both Rab11 and adaptor protein 1 (AP1), but not AP3 or AP4, are required for efficient p14 trafficking from the trans-Golgi network (TGN) to the plasma membrane. This is also the first indication of Rabs regulating adaptor proteins at the TGN for anterograde vesicle traffic, and provides a clear indication that AP1 can mediate anterograde traffic from the Golgi to the plasma membrane. I conclude that the p14 PBM functions as a novel autonomous tribasic Golgi export signal by directing interaction with activated Rab11, resulting in p14 sorting into AP1-coated vesicles at the TGN for trafficking to the plasma membrane, either directly or via endosomal recycling pathways.