Haploinsufficiency of the Sec7 Guanine Nucleotide Exchange Factor Gea1 Impairs Septation in Fission Yeast

Abstract

Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/−), the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/− cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/− mutant strain. Importantly, gea1+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

Ypt4andlvs1regulate vacuolar size and function inSchizosaccharomyces pombe

“… 26 Furthermore, membrane trafficking pathways have been implicated in control of vacuolar size in yeast. 27 , 28 Therefore, we next analyzed general endocytic and secretory trafficking pathways in cells lacking ypt4 or lvs1 . Endocytosis was quantitatively analyzed by monitoring FM4–64 uptake using flow cytometry.…”

“…Image J was used to quantify vacuole size as described previously. 27 Results are reported as the mean ± standard error of the mean (SEM).…”

“…FM4–64 staining was performed as described previously. 17 , 27 Briefly, cultures (WT, ypt4Δ, lvs1Δ , and ypt4-YFP and lvs1-YFP overexpression, as indicated) were resuspended in fresh YES media containing 32 µM FM4–64 (ThermoFisher Scientific) and incubated at 30°C for 20 minutes. After washing, the cells were resuspended in fresh YES media and incubated at 30°C for 30 min to facilitate delivery of FM4–64 to the vacuole.…”

“…For vacuolar fusion and recovery assays, WT, ypt4Δ , and lvs1Δ cells were stained with FM4–64, as described above. To induce fusion, instead of resuspending cells in fresh YES media, cells were resuspended in dH 2 O and incubated for 90 min, 17 , 27 followed by washing. Cells that underwent recovery were incubated for an additional 90 min at 30°C before imaging and vacuolar quantification as described above.…”

“…Secretion of acid phosphatase activity was assayed as described previously. 27 , 28 Equal numbers of WT, ypt4Δ , and lvs1Δ cells were resuspended in fresh YES media and incubated at 30°C for the indicated times. At each timepoint, a media sample was collected for acid phosphatase secretion analysis and immediately subjected to centrifugation at 25000 × g for 1 min.…”