Expression of either AtVTI1a or AtVTI1b allows yeast cells to grow in the absence of Vti1p, but only AtVTI1a functions in TGN-to-PVC traffic. (A) Growth of the vti1Δ GAL1-VTI1 strain (FvMY6/pFvM16) alone or expressing AtVTI1a or AtVTI1b on plates containing galactose (Gal) or glucose (Glc). The vti1Δ GAL1-VTI1 strain could not grow on glucose after the expression of VTI1 was shut off, but expression of either AtVTI1a or AtVTI1b supported growth. (B and C) CPY traffic in wild-type, vti1-1 (FvMY7) (B) and vti1-11 (C) cells (FvMY21) alone (−) or expressing AtVTI1a or AtVTI1b. Cells were grown at 24°C, preincubated at 36°C for 15 min, pulse labeled for 10 min, and chased for 30 min at 36°C. CPY was immunoprecipitated from cellular extracts (I) and extracellular fractions (E) and analyzed by SDS-PAGE and autoradiography. The TGN-to-PVC traffic block in vti1-1 cells (FvMY7) was suppressed by AtVTI1a expression, as indicated by the presence of vacuolar mCPY, but not by AtVTI1b expression. (C) vti1-11 cells (FvMY21) accumulated p1CPY because of a block in retrograde traffic to the cis-Golgi. Expression of either AtVTI1a or AtVTI1b reduced the amount of p1CPY that accumulated.