Figure 4.

Atg9 colocalizes with Arp2. (A) Atg9 and Arp2 were tracked in a single live cell in wild-type (IRA033) and atg1Δ (IRA037) cells expressing chromosomally tagged Atg9-3xGFP and TPI promoter-driven Arp2-3xDsRed, and Arp2 and chromosomally tagged Vrg4-GFP were monitored in wild-type cells (IRA039). Mid-log phase cells were analyzed by simultaneous two-color imaging combined in 4D (x,y,z,t). The RFP and GFP images were collected simultaneously with a time interval between Z-stacks acquisitions of 20 s for 120-s duration. A collapsed series of Z-sections is shown at the 60 s time point. Scale bar, 2 μm. (B) Still frames from a collapsed series of Z-sections and individual Z-sections at a single focal plane from a different cell demonstrate Atg9-3xGFP and Arp2-3xDsRed colocalization. The single Z-section images show the magnified view of the boxed area. (C) Quantification of the number of Atg9-3xGFP puncta colocalized with Arp2-3xDsRed in the wild-type and atg1Δ strains, and the number of Vrg4-GFP dots colocalized with Arp2-3xDsRed (n = 100). The number of Atg9 dots that colocalized with Arp2 in the wild-type strain per cell was set to 100%. (D) Arp2-3xDsRed is functional. arp2-1 cells expressing Arp2-DsRed or empty vector were grown to mid-log phase at 24°C, and then they were incubated for 3 h either at 24 or 37°C. Precursor Ape1 maturation was monitored by Western blot. The positions of precursor and mature Ape1 are indicated. (E) Atg9-3xGFP is functional. Wild-type cells or atg9Δ cells expressing Atg9-3xGFP or empty vector were grown to mid-log phase, and then they were examined by Western blot as described in C.