FIGURE 1:

Overexpression of Arl1 or Imh1 restores endosome-to-TGN transport and suppresses temperature-sensitive growth defects in ypt6Δ cells. (A) Overexpression of Arl1 or Imh1, but not Syt1 or Arl3, restores Snc1 and Tlg1 transport defects in ypt6Δ cells. Fluorescently tagged Snc1 or Tlg1 was coexpressed with Arl3, Syt1, Arl1, or Imh1 in ypt6Δ cells. Live cells were observed in mid–log phase using fluorescence microscopy (scale bar, 5 μm). (B) Overexpression of Arl1 or Imh1 suppresses the temperature-sensitive growth defects in ypt6Δ cells. ypt6Δ cells transformed with Ypt6, different forms (wild-type [WT], constitutively active [QL], and constitutively inactive [TN]) of Arl1 or Arl3, and Imh1 were serially diluted 10-fold as indicated, spotted on plates, and incubated at 30 or 37°C. (C) Arl1 and Imh1 restore the distribution of GFP-Snc1 in ypt6Δ cells. WT and ypt6Δ cells were transformed with Arl1 and Imh1. Lysates of spheroplasts from cells were fractionated by sucrose gradient (20–60%) centrifugation. Proteins in the fractions were precipitated, separated by SDS–PAGE, and analyzed by immunoblotting. Pma1 (plasma membrane marker), GFP-Snc1, and Emp47 (early Golgi marker) were identified with specific antibodies and detected using an ECL system. Gradient fractions were numbered from the top.