BFA sends proteins back

O bserving a step backward brought Jon Yewdell a big step forward in understanding protein traffi cking pathways in the cell. As a newly minted assistant professor at the Wistar Institute, Yewdell was examining where in the cell the infl uenza A virus (IAV) hemagglutin (HA) trimerized. Using monoclonal antibodies (mAbs) specifi c for monomers or trimers, he had found by 1985 that monomers localized to the en-doplasmic reticulum (ER), whereas trimers were fi rst detected in the Golgi complex without a trace of ER staining (Yewdell et al., 1988). And there the trimers might have stayed except for a chance conversation. Hearing Yewdell's seminar at Case Western Reserve University, Alan Tartakoff urged him to read Akira Takatsuki's paper showing that the drug Brefeldin A (BFA) blocked transport from the ER (Misumi et al., 1986). Yewdell wrote to Takatsuki, who generously sent a glassine envelope with a few milligrams of powder. Recruited to the National Institutes of Health in 1987, Yewdell forgot about BFA until the envelope tumbled out of a lab notebook while he was unpacking. He treated IAV-infected cells with BFA, and observed for the first time ER staining with trimer-specific mAbs. " After stumbling around in the dark for a suitable period, " says Yewdell, he eventually performed an intricate pulse-chase experiment in which he fi rst blocked HA egress from the ER with BFA and then removed the BFA in the presence of cycloheximide to prevent new HA synthesis. Now, the trimers completely cleared the ER and moved to the Golgi on their way to the cell surface. When Yewdell added back BFA during the chase, HA trimers magically reappeared in the ER. " I pretty much knew that I had discovered retrograde transport, " he says. Retrograde traffi cking from the Golgi to ER had been suggested both for retrieval of ER resident proteins (Pelham, 1988) and recycling of lipids (Wieland et al., 1987). But no techniques could isolate this pathway from anterograde traffi c. Yewdell needed a method to prove that BFA was not holding up forward traffi c but instead triggering reverse traffi c. To get the experiment going he therefore needed to conditionally trap a marker protein in the ER without using BFA. So he recruited his friend Robert Doms (then a post-doctoral student in a neighboring lab). Doms had been studying a vesicular stomatitis virus temperature-sensitive mutant that, at 40°C, produces …