Fig. 6. Pex35 and peroxisomes control Arf1 distribution and function.

(A) Arf1 distribution is impaired in pex35 mutants. While Arf1-RFP is localized in punctate structures (potentially including Golgi and mitochondria) and partially colocalizes to peroxisomes (white arrowheads) in wild-type (WT) strains, deletion or overexpression (OE) of PEX35 results in re-distribution to other internal membranes and loss of punctate localization. Scale bar: 5 μm. (B) Synthetic effects of PEX35 and ARF1 mutations. Pex3-GFP was used as a marker to quantify the peroxisome abundance in combinations of Δarf1 and WT, Δpex35 or OE-PEX35. Scale bar: 5 μm. (C) Quantification of peroxisomal content reveals that deletion of ARF1 aggravates the Δpex35 phenotype of reduced peroxisome number. Notably, Δarf1 deletion compensated for the effect of PEX35 overexpression on peroxisome abundance. Error bars indicate s.e.m., n>197. ***P<0.01 compared with WT (t-test). (D) Protein secretion analysis of PEX35 and ARF1 mutants. Images (left; the levels of secretion of Kar2 are indicated) and quantification (right) of a western blot of Kar2 secreted from six colony repeats per strain. Induction of the UPR (by deletion of SPF1) or loss of peroxisomes (by deletion of PEX3), as well as overexpression of GFP-Pex35 or ARF1 significantly reduced secretion levels. Deletion of PEX35 compensated for the effect of ARF1 overexpression. Error bars indicate s.d., n=6. *P<0.1 (not significant); ***P<0.01 (t-test). (E) Deletion of PEX3 affects cellular Arf1-RFP distribution. InΔpex3 cells, Arf1-RFP loses its localization to defined puncta. Scale bar: 5 μm.