Fig. 4.

Sugar transporter activity is essential for Spin's role in the regulation of autophagic lysosome reformation. (A) Carbohydrates accumulate in spin knockdown cells under prolonged starvation. NRK (CFP-LC3) cells were transfected with NS- or spin-RNAi. After 2 d, cells were starved for 18 h and subjected to periodic acid-Schiff (PAS) staining. (Scale bars, 5 μm.) Arrows indicate PAS-positive structures. Histogram shows the percentage of PAS-positive structures from multiple experiments counting a total of 100 cells, with mean and SD shown. (B) The alignment of the spin amino acid sequence from representative species shown in standard single letter code. The conserved glutamic acid residue (E) which is essential for sugar transporter activity is highlighted in red. (C) NRK cells were transfected with RNAi against spin. Two days after transfection, cells were retransfected with RNAi and vector/Lamp1-YFP, hSpin-CFP-wt/Lamp1-YFP or hspin-CFP-mutant (spinE217K)/Lamp1-YFP. Twenty-four hours after the second transfection, cells were starved for 10 h and then observed by confocal microscopy. (Scale bar, 5 μm.)