Figure 14.

Proposed trafficking of wt-hCgB and Δcys-hCgB after transfection and vaccinia expression. Transfection (left): Both recombinant (filled squares) and endogenous (open squares) granins are present in the TGN. wt-hCgB and Δcys-hCgB are sorted upon exit from the TGN into ISG. Sorting of wt-hCgB (top left, squares with semicircle) is accomplished by the formation of aggregates and by the disulfide-bonded loop. Sorting to ISG of Δcys-hCgB, which lacks the disulfide-bonded loop (middle left, plain squares) is accomplished by its coaggregation with endogenous granins bearing the disulfide-bonded loop. A minor amount of Δcys-hCgB exits from the TGN into CV, probably due to formation of homotypic aggregates lacking endogenous granins. Vaccinia system (right): Recombinant hCgB is present in the TGN whereas endogenous granins are absent due to the shut-off of host cell protein synthesis. After single infection, wt-hCgB (top right, filled squares with semicircle) is mostly sorted to ISG. The small amount exiting from the TGN into CV is due to factors involved in sorting, which become rate-limiting under shut-off. In contrast, Δcys-hCgB lacking the disulfide-bonded loop (middle right, plain filled squares) exits from the TGN completely into CV after single infection. Thus, due to the absence of loop-bearing endogenous granins, sorting of Δcys-hCgB to ISG cannot be rescued by coaggregation. However, after double infection, i.e., expression of Δcys-hCgB together with wt-hCgB-EGFP (filled squares with semicircle and triangle), sorting of Δcys-hCgB to ISG is rescued by coaggregation with loop-bearing wt-hCgB-EGFP, and only a minor portion exits into CV. Thickness of arrows reflects various amounts of trafficking proteins; dashed arrow, absence of traffic.