Haploinsufficiency of the Sec7 Guanine Nucleotide Exchange Factor <em>Gea1</em> Impairs Septation in Fission Yeast

<div><p>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 <em>gea1</em> (<em>gea1</em>+/−), the <em>Schizosaccharomyces pombe</em> ortholog of <em>GBF1</em>. The haploinsufficient <em>gea1</em>+/− 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 <em>gea1+/−</em> 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 <em>gea1</em>+/− mutant strain. Importantly, <em>gea1</em>+/− 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 <em>gea1</em>+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate <em>gea1</em> in regulation of septum breakdown and establish <em>S. pombe</em> as a model system to explore GBF1/GEA function in cytokinesis.</p> </div

Organellar morphology in <i>gea1+/−</i> cells.

<p>A. Wild-type and <i>gea1+/−</i> cells were stained with 5 µM BODIPY FL C₅-ceramide to label the Golgi or with 32 µM FM4-64 to label the vacuole. Staining was visualized by fluorescence microscopy. Scale bar, 10 µM. B. The pixel area associated with the largest vacuole was measured using Image J for individual wild-type (n = 262) and <i>gea1+/−</i> (n = 225) cells. The percentage of cells with vacuolar sizes of the indicated ranges was plotted using SigmaPlot. C. Wild-type (A) and <i>gea1</i>+/− cells (B) were subjected to transmission electron microscopy to visualize membranous structures. Representative images show flat ribbon-like structures consistent with Golgi membranes (insets) that appear similar in wild-type and <i>gea1+/−</i> cells. N, nucleus. Scale bar, 500 nm. D. Cells were labeled with FM4-64, followed by incubation in H₂O for 90 min to induce vacuolar fusion. Scale bar, 10 µM.</p

Primers used in this study.

<p>Primers used in this study.</p

COP-I-dependent transport is impaired in <i>gea1+/−</i> cells.

<p>A. Wild-type and <i>gea1+/−</i> cells were transformed with pDUAL-YFH1c-<i>arf1</i> or pDUAL-YFH1c-<i>arf6</i> and imaged by fluorescence microscopy. Scale bar, 10 µM. B. Wild-type and <i>gea1+/−</i> cells were transformed with pDUAL-YFH1c-<i>sac11</i> (SPBC19F5.03) or pDUAL-YFH1c-<i>sac12</i> (SPAC3C7.01c) and imaged by fluorescence microscopy. Scale bar, 10 µM. C. Localization of sac11-YFP in wild-type (n = 207) and <i>gea1+/−</i> cells (n = 110) was scored as ER (surrounding the cell cortex and nuclear envelope), Golgi (punctate in the cytoplasm), or mixed. Sac11-YFP was predominately found in the ER in wild-type cells and in the Golgi and mixed in <i>gea1+/−</i> cells. D. Localization of sac12-YFP in wild-type (n = 204) and <i>gea1+/−</i> cells (n = 147) was scored as ER, Golgi, or mixed. Sac11-YFP was predominately found in the Golgi in both wild-type and <i>gea1+/−</i> cells. Error bars represent the mean ± SD from 3 independent experiments.</p

<i>Gea1+/−</i> cells exhibit alterations in septum number and morphology.

<p>A. Wild-type and <i>gea1</i>+/− cells were stained with calcofluor white to visualize septa and imaged by fluorescence microscopy. Scale bar, 10 µM. B. Quantification of A. Cells were scored as having abnormal septa if multiple septa were present, or if the septum was mislocalized, abnormally thick, or forked. Error bars represent mean ± SD from 3 independent experiments. C. Wild-type and <i>gea1</i>+/− cells were subjected to transmission electron microscopy to visualize septum defects. Representative images of multi-septated cells and septa with morphological abnormalities are shown. Scale bar, 1 µm.</p

Sensitivity of <i>gea1+/−</i> cells to BFA can be rescued by overexpression of gea1p.

<p>A. <i>Gea1+/−</i> cells were transformed with pDUAL-YFH1c-<i>gea1</i> to drive expression of gea1-YFP. Spinning-disc confocal fluorescence microscopy revealed that gea1-YFP localized to punctate structures in the cytoplasm that colocalized with the Golgi-specific stain BODIPY® TR C₅-ceramide. Scale bar, 14 µM. B. Wild-type and <i>gea1+/−</i> cells expressing endogenous gea1 tagged with GFP were imaged by fluorescence microscopy. The gea1-GFP protein localizes to punctate cytoplasmic structures. Scale bar, 10 µM. C. Wild-type, <i>gea1</i>+/−, and <i>gea1+/−</i> cells transformed with pDUAL-YFH1c-<i>gea1</i> (<i>gea1+/−</i> + gea1-YFP) cells were subjected to 10-fold serial dilution and spotted on YES media and YES +10 µg/mL BFA. Plates were incubated at 30°C for 3 days. D. Equal numbers (5×10⁴ cells) of wild-type, wild-type (WT) + pDUAL-YFH1c (empty vector, WT + EV), wild-type + pDUAL-YFH1c-<i>gea1</i> (WT + gea1), <i>gea1+/−</i>, <i>gea1+/−</i> + pDUAL-YFH1c, (gea1+/<i>−</i> + EV), and <i>gea1+/−</i> + pDUAL-YFH1c-<i>gea1</i> (gea1+/− + gea1) cells were incubated in YES media containing the indicated concentrations of BFA at 30°C. After 24 h, cell density was assessed by monitoring the optical density of the cultures at 600 nm. The results are reported at as a percentage of the density of control untreated cultures. Error bars represent mean ± SE (n = 3). Restoration of gea1p expression suppressed the BFA sensitivity of <i>gea1+/−</i> cells.</p