In yeast, mitochondrial department and fusion are regulated during development, sporulation

In yeast, mitochondrial department and fusion are regulated during development, sporulation and mating, yet the systems controlling these actions are unknown. EcoRV-BamHI fragment from pHS1 was put into pDH9, which bears 5 and 3 untranslated parts of (something order Vargatef special from S. Michaelis), forming pHS2. To integrate at chromosomal from pHS2 was changed into stress SM1235, which bears ( Michaelis and Herskowitz 1988). Stress YHS2, which consists of stress YHS1 was acquired by crossing YHS2 to SM1227 ( Michaelis and Herskowitz 1988). Stress 1002 (stress 194 (something special from E. Schweizer) or stress JSY1361 ( Otsuga et al. 1998) showed the course IV mutation was centromere connected, situated on chromosome XII, and allelic to and were constructed by PCR-mediated gene replacement as described ( Lorenz et al. 1995) into strains BY4733 and BY4744 ( Brachmann et al. 1998). For plasmid pRS303 ( Sikorski and Hieter 1989) and for we used plasmid pRS400 ( Brachmann et al. 1998). MATa strain YHS27 and MAT strain YHS23 were constructed by crossing MATa strain YHS19 to MAT strain YHS22. Mitochondria in the disruption strains were visualized using pHS12, a plasmid made up of gene with a NotI site immediately preceding its termination codon was PCR amplified from yeast genomic DNA and subcloned into pAA3, a plasmid which contains the HA epitope with a NotI site at its NH2 terminus (Aiken, A., unpublished data), forming pDNM1-HA (pHS14). DNM1-GFP plasmid pHS20 was constructed as described above except that Rabbit Polyclonal to CRHR2 pAA1, a plasmid which contains GFP with a NotI site at its NH2 terminus (Aiken, A., unpublished data), was used instead of pAA3. To form pHS15, coding sequences were PCR amplified from pHS14 with 50 bp of flanking sequences homologous to the promoter in pRS314GU ( Nigro et al. 1992). The fragment and linearized pRS314GU were cotransformed into yeast and pGAL1-DNM1-HA (pHS15) was formed by homologous recombination ( Oldenburg et al. 1997). pGAL1-DNM1-GFP (pHS40) was constructed as described for pHS15 except that pHS20 was used instead of pHS14. Quantitation of Dnm1p-GFP Localization cells carrying pGAL1-DNM1-GFP were incubated in galactose media for 1C2 h, stained with MitoTracker Red CMXRos (Molecular Probes). 12 cells were examined by fluorescence microscopy and the mitochondrially associated Dnm1p-GFP dots (82 total) were assigned to one of two locations: (a) the end of a tubule (50 dots), or (b) the side of a tubule (32 dots). The end of a tubule was defined as when the center of a Dnm1p-GFP dot was located within 0.15 m from the end. The average length of the mitochondrial tubules was estimated to be 2.7 1.9 m and the diameter 0.3 m (= 44). order Vargatef We calculate that the side of the tubule represents 89% order Vargatef of the mitochondrial surface area and the remainder (11%) represents the end. Results and Discussion We screened for yeast mutants defective in mitochondrial shape using a novel strategy in which mitochondria are visualized by the green fluorescent protein (GFP) and mutants were isolated by micromanipulation. GFP was fused to the presequence (residues 1C21) of mitochondrial cytochrome oxidase subunit IV (COX4; Pon and Schatz 1991). When expressed order Vargatef in yeast, COX4-GFP targets the mitochondrial matrix, and mitochondria were visible by fluorescence microscopy. We integrated the COX4-GFP gene at the nonessential locus ( Michaelis and Herskowitz 1988), which made fluorescence intensity uniform among cells and enabled efficient screening. After mutagenesis, individual cells.