Fig. S2. Subcellular localization of GFP-CHC-1. (A-C) GFP-CHC-1 is localized to the plasma membrane and punctate structures in oocytes (A, B). As the most mature oocyte began to ovulate, GFP-CHC-1 lost its punctate localization and appeared diffuse (arrow). (C) Nomarski image. (D-F) Gonads expressing GFP-CHC-1 or GFP-CHC-1
(b1025) were dissected and observed by wide-field deconvolution fluorescence microscopy. GFP-CHC-1, and to a lesser degree GFP-CHC-1
(b1025), were localized to the plasma membrane when expressed in a wild-type strain (D, E). When GFP-CHC-1
(b1025) was expressed in
chc-1(
b1025ts) mutants, where it cannot form mixed oligomers with wild-type CHC-1, it lacked most plasma membrane localization (F). It is not clear if the residual punctate appearance of the mutant protein reflects association with internal membranes or some kind of protein aggregation. (Scale bars, 10 um.) (G-M) GFP-CHC-1
(b1025) is unstable at high temperature. (G-L) GFP-CHC-1 (G, J) or GFP-CHC-1
(b1025) (H, I, K, L) were expressed in the intestine under the control of the
vha-6 promoter. L4 larvae were incubated at 15C (G-I) or 25C (J-L) for 24 h and observed by confocal laser scanning microscopy. The images in G, H, J, and K were taken using the same conditions. I and L are overexposed images of H and K, respectively. (Scale bars, 10 um.) GFP-CHC-1 was localized predominantly to the apical membrane and punctate structures in the cytoplasm at 15C and 25C (G, J). On the other hand, the expression level of GFP-CHC-1
(b1025) was quite low even at the permissive temperature (H, I) and was reduced further at the restrictive temperature (K, L). (M) GFP-CHC-1
(b1025) is unstable at restrictive temperature. Worms expressing GFP-CHC-1 or GFP-CHC-1
(b1025) in the intestine were incubated at 15C or 25C for 24 h or at 30C for 10 min or 120 min and subjected to Western blotting. GFP fusions and RME-1 were detected using anti-GFP antibody and anti-RME-1 antibody, respectively. The level of GFP-CHC-1
(b1025) was reduced in a temperature-dependent manner compared with GFP-CHC-1.