Gag3p, an outer membrane protein required for fission of mitochondrial tubules

Abstract

Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

Figure 1

Suppressing mutations prevent mitochondrial fragmentation in the mdm17 mutant. Wild-type (MYY290; left), mdm17 (MYY971; center), and suppressed mdm17 (MYY993) cells were grown at 37°C on YPD-medium, stained with DASPMI, and visualized by fluorescence microscopy. Bar, 2 μm.

Figure 3

GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.

Figure 3

GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.

Figure 3

GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.

Figure 2

gag Mutants display abnormal mitochondrial morphology. Wild-type (MYY1200), gag1 (MYY2033), gag2 (MYY2029), or gag3 (MYY2031) cells with GFP-labeled mitochondria were cultured on YPD-medium and examined by fluorescence microscopy. Two representative cells are shown for each mutant strain. Bar, 2 μm.

Figure 4

Gag3p is a peripheral protein of the mitochondrial outer membrane. A, Cells expressing HA-tagged Gag3p (MYY2016) or myc-tagged Dnm1p (MYY1202) were grown on semisynthetic lactate medium (Daum et al. 1982). Cells were homogenized and subcellular fractions were isolated by differential centrifugation. The homogenate (hom), low-speed pellet (LSP), mitochondrial (mito), intermediate-speed pellet (ISP), high-speed pellet (HSP), and the cytosolic fractions (cyto) were analyzed by SDS-PAGE and immunoblotting to detect Gag3p, Dnm1p, the mitochondrial outer membrane protein OM45, and the cytosolic protein glucose-6-phosphate dehydrogenase (G6PDH). Each lane contained 10 μg total protein. B, Trypsin treatment of mitochondria. Isolated mitochondria containing HA-tagged Gag3p were treated with trypsin in the presence (+) or absence (−) of deoxycholate detergent and analyzed by SDS-PAGE and immunoblotting. Samples were tested for the presence of Gag3p, the outer membrane protein Tom70p, the intermembrane space protein cytochrome b₂, and the mitochondrial matrix protein Mas2p. C, Gag3p is peripherally associated with mitochondrial membranes. Isolated mitochondria containing HA-tagged Gag3p were treated with 1 M NaCl, 0.1 M Na₂CO₃, or 8 M urea. Mitochondria were centrifuged, and the resulting pellet (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting for Gag3p and OM45.

Figure 5

Localization of Dnm1p in gag mutant cells. Wild-type (MYY290), gag2 (MYY981), and gag3 (MYY2017) cells expressing GFP-tagged Dnm1p were cultured on minimal glucose medium, stained with MitoTracker Red CMXRos, and analyzed by fluorescence microscopy. Pseudocolor was added to the digitized image. GFP-labeling is shown in green, MitoTracker is shown in red, and the merged images are shown on the right. Bar, 2 μm.