ADP-ribosylation factor 1-independent protein sorting and export from the trans-Golgi network.
Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from the trans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using an in vitro reconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) as well as under conditions known to negatively regulate apical transport in vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPgammaS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Mark A
MA
Ellis
Mark T
MT
Miedel
Christopher J
CJ
Guerriero
Ora A
OA
Weisz
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006367:HeLa Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D011506:Proteins / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011506:Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D020823:ADP-Ribosylation Factor 1