Three-dimensional tumor visualization of invasive breast carcinomas using whole-mount serial section histopathology: implications for tumor size assessment.

PURPOSE: Linear tumor size (T-size) estimated with conventional histology informs breast cancer management. Previously we demonstrated significant differences in margin and focality estimates using conventional histology versus digital whole-mount serial sections (WMSS). Using WMSS we can measure T-size or volume. Here, we compare WMSS T-size with volume, and with T-size measured conventionally. We also compare the ellipsoid model for calculating tumor volume to direct, WMSS measurement. METHODS: Two pathologists contoured regions of invasive carcinoma and measured T-size from both WMSS and (simulated) conventional sections in 55 consecutive lumpectomy specimens. Volume was measured directly from the contours. Measurements were compared using the paired t-test or Spearman's rank-order correlation. A five-point 'border index' was devised and assigned to each case to parametrize tumor shape considering 'compactness' or cellularity. Tumor volumes calculated assuming ellipsoid geometry were compared with direct, WMSS measurements. RESULTS: WMSS reported significantly larger T-size than conventional histology in the majority of cases [61.8%, 34/55; means = (2.34 cm; 1.99 cm), p < 0.001], with a 16.4% (9/55) rate of 'upstaging'. The majority of discordances were due to undersampling. T-size and volume were strongly correlated (r = 0.838, p < 0.001). Significantly lower volume was obtained with WMSS versus ellipsoid modeling [means = (1.18 cm3; 1.45 cm3), p < 0.001]. CONCLUSIONS: Significantly larger T-size is measured with WMSS than conventionally, due primarily to undersampling in the latter. Volume and linear size are highly correlated. Diffuse tumors interspersed with normal or non-invasive elements may be sampled less extensively than more localized masses. The ellipsoid model overestimates tumor volume.

Factors influencing survival among patients with HER2-positive metastatic breast cancer treated with trastuzumab.

PURPOSE: We have limited capability to predict survival among patients treated for metastatic HER2-positive breast cancer. Further research is warranted to identify significant prognostic and predictive factors. METHODS: We identified all HER2-positive metastatic breast cancer patients receiving trastuzumab at the Sunnybrook Odette Cancer Centre (SOCC) from 1999 to 2013 through the Cancer Care Ontario (CCO) Registry (n = 256) and selected patients with available pathology reports (n = 154). A retrospective review was completed documenting clinical, pathologic, and laboratory characteristics at the time of first trastuzumab therapy and survival outcomes. Cox proportional hazards regression models were used to identify prognostic factors for overall survival (OS) (primary endpoint) and failure-free survival (FFS), adjusted for the known prognostic factors of the presence of CNS metastases and the presence of ≥ 2 distant metastatic sites. RESULTS: A multivariable model identified older age [hazard ratio (HR) 1.18/decade, 95% confidence interval (CI) 1.02-1.37)], increased platelet-to-lymphocyte ratio (PLR) (HR 1.75/log-unit, 95% CI 1.25-2.46), increased serum alkaline phosphatase (ALP) (HR 1.87/log-unit, 95% CI 1.41-2.49), and ER positivity (HR 0.63, 95% CI 0.42-0.96) as significant prognostic factors for OS after adjusting for the presence of CNS metastasis (HR 3.19, 95% CI 1.59-6.38) and the presence of ≥ 2 distant metastatic sites (HR 2.10, 95% CI 1.19-3.70). PLR (HR 1.54/log-unit, 95% CI 1.12-2.12) was the only prognostic factor associated with FFS after adjusting for CNS and ≥ 2 distant metastatic sites. CONCLUSION: Older age, increased PLR, and ALP were identified as poor prognostic factors and ER positivity as a favorable prognostic factor for OS after adjusting for the presence of CNS metastasis and the presence of number of ≥ 2 distant metastatic sites. Increased PLR was a poor prognostic factor for both OS and FFS, and warrants further investigation into its prognostic ability amongst patients with HER2-positive metastatic breast cancer.

Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy.

Polymorphonuclear (PMN) leucocytes participate in acute inflammatory pathologies such as acute respiratory distress syndrome (ARDS) following traumatic injury and shock, which also activates the coagulation system systemically. Trauma can prime the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex for an enhanced respiratory burst, but the relative role of various priming agents in this process remains incompletely understood. We therefore set out to identify mediators of PMN priming during coagulation and trauma-shock and determine whether PMN reactive oxygen species (ROS) generated in this manner could influence organ injury and coagulation. Initial experiments demonstrated that PMN are primed for predominantly extracellular ROS production by products of coagulation, which was abrogated by CD88/C5a receptor(C5aR) inhibition. The importance of this was highlighted further by demonstrating that known PMN priming agents result in fractionally different amounts of extracellular versus intracellular ROS release depending on the agent used. Plasma from trauma patients in haemodynamic shock (n = 10) also primed PMN for extracellular ROS in a C5a-dependent manner, which correlated with both complement alternative pathway activation and thrombin generation. Furthermore, PMN primed by preincubation with products of blood coagulation directly caused loss of endothelial barrier function in vitro that was abrogated by C5aR blockade or NADPH oxidase inhibition. Finally, we show in a murine model of trauma-shock that p47phox knock-out (KO) mice with PMN incapable of generating ROS were protected from inflammatory end-organ injury and activated protein C-mediated coagulopathy. In summary, we demonstrate that trauma-shock and coagulation primes PMN for predominantly extracellular ROS production in a C5a-dependent manner that contributes to endothelial barrier loss and organ injury, and potentially enhances traumatic coagulopathy.

Cost-effectiveness analysis of whole-mount pathology processing for patients with early breast cancer undergoing breast conservation.

BACKGROUND: Obtaining accurate histopathologic detail for breast lumpectomy specimens is challenging because of sampling and loss of three-dimensional conformational features with conventional processing. The whole-mount (wm) technique is a novel method of serial pathologic sectioning designed to optimize cross-sectional visualization of resected specimens and determination of margin status. METHODS: Using a Markov chain cohort simulation cost-effectiveness model, we compared conventional processing with wm technique for breast lumpectomies. Cost-effectiveness was evaluated from the perspective of the Canadian health care system and compared using incremental cost-effectiveness ratios (icers) for cost per quality-adjusted life-year (qaly) over a 10-year time horizon. Deterministic and probabilistic sensitivity analyses were performed to test the robustness of the model with willingness-to-pay (wtp) thresholds of $0-$100,000. Costs are reported in adjusted 2014 Canadian dollars, discounted at a rate of 3%. RESULTS: Compared with conventional processing, wm processing is more costly ($19,989 vs. $18,427) but generates 0.03 more qalys over 10 years. The icer is $45,414, indicating that this additional amount is required for each additional qaly obtained. The model was robust to all variance in parameters, with the prevalence of positive margins accounting for most of the model's variability. CONCLUSIONS: After a wtp threshold of $45,414, wm processing becomes cost-effective and ultimately generates fewer recurrences and marginally more qalys over time. Excellent baseline outcomes for the current treatment of breast cancer mean that incremental differences in survival are small. However, the overall benefit of the wm technique should be considered in the context of achieving improved accuracy and not just enhancements in clinical effectiveness.

Long-term results of screening with magnetic resonance imaging in women with BRCA mutations.

BACKGROUND: The addition of breast magnetic resonance imaging (MRI) to screening mammography for women with BRCA mutations significantly increases sensitivity, but there is little data on clinical outcomes. We report screening performance, cancer stage, distant recurrence rate, and breast cancer-specific mortality in our screening study. METHODS: From 1997 to 2009, 496 women aged 25 to 65 years with a known BRCA1/2 mutation, of whom 380 had no previous cancer history, were enrolled in a prospective screening trial that included annual MRI and mammography. RESULTS: In 1847 screening rounds, 57 cancers were identified (53 screen-detected, 1 interval, and 3 incidental at prophylactic mastectomy), of which 37 (65%) were invasive. Sensitivity of MRI vs mammography was 86% vs 19% over the entire study period (P<0.0001), but was 74% vs 35% from 1997 to 2002 (P=0.02) and 94% vs 9% from 2003 to 2009 (P<0.0001), respectively. The relative sensitivities of MRI and mammography did not differ by mutation, age, or invasive vs non-invasive disease. Of the incident cancers, 97% were Stage 0 or 1. Of 28 previously unaffected women diagnosed with invasive cancer, 1 BRCA1 mutation carrier died following relapse of a 3 cm, node-positive breast cancer diagnosed on her first screen at age 48 (annual breast cancer mortality rate=0.5%). Three patients died of other causes. None of the 24 survivors has had a distant recurrence at a median follow-up of 8.4 years since diagnosis. CONCLUSION: Magnetic resonance imaging surveillance of women with BRCA1/2 mutations will detect the majority of breast cancers at a very early stage. The absence of distant recurrences of incident cancers to date is encouraging. However, longer follow-up is needed to confirm the safety of breast surveillance.

A randomized, placebo-controlled trial (NCIC CTG MAP.2) examining the effects of exemestane on mammographic breast density, bone density, markers of bone metabolism and serum lipid levels in postmenopausal women.

We hypothesized that exemestane (EXE) would reduce mammographic breast density and have unique effects on biomarkers of bone and lipid metabolism. Healthy postmenopausal women were randomized to EXE (25 mg daily) or placebo (PLAC) for 12 months and followed for a total of 24 months. The primary endpoint was change in percent breast density (PD) between the baseline and 12-month mammograms and secondary endpoints were changes in serum lipid levels, bone biomarkers, and bone mineral density (BMD). Ninety-eight women were randomized (49 to EXE; 49 to PLAC) and 65 had PD data at baseline and 12 months. Among women treated with EXE, PD was not significantly changed from baseline at 6, 12, or 24 months and was not different from PLAC. EXE was associated with significant percentage increase from baseline in N-telopeptide at 12 months compared with PLAC. No differences in percent change from baseline in BMD (lumbar spine and femoral neck) were observed between EXE and PLAC at either 12 or 24 months. Patients on EXE had a significantly larger percent decrease in total cholesterol than in the PLAC arm at 6 months and in HDL cholesterol at 3, 6, and 12 months. No significant differences in percent change in LDL or triglycerides were noted at any time point between the two treatment arms. EXE administered for 1 year to healthy postmenopausal women did not result in significant changes in mammographic density. A reversible increase in the bone resorption marker N-telopeptide without significant change in bone specific alkaline phosphatase or BMD during the 12 months treatment period and 1 year later was noted. Changes in lipid parameters on this trial were modest and reversible.

Adiposity changes after a 1-year aerobic exercise intervention among postmenopausal women: a randomized controlled trial.

OBJECTIVE: We examined the effects of an aerobic exercise intervention on adiposity outcomes that may be involved in the association between physical activity and breast cancer risk. DESIGN: This study was a two-centre, two-armed, randomized controlled trial. The 1-year-long exercise intervention included 45 min of moderate-to-vigorous aerobic exercise five times per week, with at least three of the sessions being facility based. The control group was asked not to change their activity and both groups were asked not to change their diet. SUBJECTS: A total of 320 postmenopausal, sedentary, normal weight-to-obese women aged 50-74 years who were cancer-free, nondiabetic and nonhormone replacement therapy users were included in this study. MEASUREMENTS: Anthropometric measurements of height, weight and waist and hip circumferences; dual energy X-ray absorptiometry measurements of total body fat; and computerized tomography measurements of abdominal adiposity were carried out. RESULTS: Women in the exercise group exercised a mean of 3.6 days (s.d.=1.3) per week and 178.5 min (s.d.=76.1) per week. Changes in all measures of adiposity favored exercisers relative to controls (P<0.001). The mean difference between groups was: -1.8 kg for body weight; -2.0 kg for total body fat; -14.9 cm(2) for intra-abdominal fat area; and -24.1 cm(2) for subcutaneous abdominal fat area. A linear trend of greater body fat loss with increasing volume of exercise was also observed. CONCLUSION: A 1-year aerobic exercise program consistent with current public health guidelines resulted in reduced adiposity levels in previously sedentary postmenopausal women at higher risk of breast cancer.

Phosphoserine/threonine-binding domains.

Phosphorylation of proteins on serine and threonine residues has traditionally been viewed as a means to allosterically regulate catalytic activity. Research within the past five years, however, has revealed that serine/threonine phosphorylation can also directly result in the formation of multimolecular signaling complexes through specific interactions between phosphoserine/threonine (pSer/Thr)-binding modules and phosphorylated sequence motifs. pSer/Thr-binding proteins and domains currently include 14-3-3, WW domains, forkhead-associated domains, and, tentatively, WD40 repeats and leucine-rich regions. It seems likely that additional modules will be found in the future. The amino acid sequences recognized by these pSer/Thr-binding modules show partial overlap with the optimal phosphorylation motifs for different protein kinase subfamilies, allowing the formation of specific signaling complexes to be controlled through combinatorial interactions between particular upstream kinases and a particular binding module. The structural basis for pSer/Thr binding differs dramatically between 14-3-3 proteins, WW domains and forkhead-associated domains, suggesting that their pSer/Thr binding function was acquired through convergent evolution.

The PX domains of p47phox and p40phox bind to lipid products of PI(3)K.

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

A randomized, placebo-controlled trial (NCIC CTG MAP1) examining the effects of letrozole on mammographic breast density and other end organs in postmenopausal women.

Mammographically detected breast density has been correlated with breast cancer risk. Breast density appears to be influenced by hormonal factors including increasing age, postmenopausal status, number of pregnancies, lower body weight, hormone replacement therapy, and tamoxifen therapy. The aromatase inhibitor letrozole profoundly reduces breast and circulating estrogen levels in postmenopausal women. We hypothesize that letrozole may reduce breast density and report here on its effects on mammographic breast density, bone mineral density (BMD), bone biomarkers, plasma hormone, and serum lipid levels. MAP1 was a multicenter, randomized, double-blind, placebo-controlled, feasibility trial in which postmenopausal women with or without prior invasive breast cancer were randomized in a 2:1 ratio of letrozole (2.5 mg daily) or placebo for 12 months and followed for a total of 24 months. Eligible women had an estimated >25% breast density on baseline mammogram. The primary endpoint was change in percent breast density (PD) between the baseline and 12-month mammograms as estimated by a computer-assisted thresholding program. Baseline and 12-month mammographic density was also assessed in a blinded manner by visual inspection. Secondary endpoints included changes in serum hormones, plasma lipid levels, bone biomarkers, and BMD. Data are available for 67 women (44 on letrozole and 23 on placebo). No significant changes in PD were noted between the treatment arms at either 12 or 24 months. No distinguishable difference in density measurements by visual inspection were noted between baseline and 12-month mammograms. A significant decrease in percentage change in T-score of the femoral neck at 12 months was noted in the letrozole arm without other significant changes in BMD parameters. Lipid values did not differ between treatment groups except for a borderline significant decrease in total cholesterol at 3 months among women treated with letrozole. Letrozole therapy was associated with a significant reduction in mean serum estradiol, estrone, and estrone sulfate levels at 12 months, but not at 24 months. A significant increase in serum IGF-1 levels was also noted in the letrozole group compared to the placebo group at both 12 and 24 months. To conclude, compared with placebo, 12 months of letrozole therapy does not appear to have a significant effect on mammographic PD. Twelve months of letrozole was associated with a decrease of uncertain clinical significance in the T-score of the femoral neck at 12 months which was reversible at 24 months with recovery of estrogen levels. Letrozole therapy was found to increase IGF-1 levels at 12 and 24 months.

Organelle inheritance in the yeast cell cycle.

Cell proliferation requires the inheritance of subcellular organelles, yet little is known of the molecular basis of this essential process. Recent microscopy studies of the yeast Saccharomyces cerevisiae have characterized the cellular distribution of mitochondria, vacuoles and elements of the endoplasmic reticulum and Golgi complex. In addition, genetic and microscopical approaches have allowed the isolation and analysis of mutants defective in the inheritance of mitochondria and vacuoles. These investigations are leading to the identification of molecular components mediating the movement of organelles into daughter cells and have revealed that the inheritance of organelles is coordinated with other events of the cell division cycle.

A role for unsaturated fatty acids in mitochondrial movement and inheritance.

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.

Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance.

Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm. These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature. To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated. Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution. Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells. Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds. The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein. Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation. These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane.

Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.

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

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.

Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein.

The mdml mutation causes temperature-sensitive growth and defective transfer of nuclei and mitochondria into developing buds of yeast cells at the nonpermissive temperature. The MDM1 gene was cloned by complementation, and its sequence revealed an open reading frame encoding a potential protein product of 51.5 kD. This protein displays amino acid sequence similarities to hamster vimentin and mouse epidermal keratin. Gene disruption demonstrated that MDM1 is essential for mitotic growth. Antibodies against the MDM1 protein recognized a 51-kD polypeptide that was localized by indirect immunofluorescence to a novel pattern of spots and punctate arrays distributed throughout the yeast cell cytoplasm. These structures disappeared after shifting mdm1 mutant cells to the nonpermissive temperature, although the cellular level of MDM1 protein was unchanged. Affinity-purified antibodies against MDM1 also specifically recognized intermediate filaments by indirect immunofluorescence of animal cells. These results suggest that novel cytoplasmic structures containing the MDM1 protein mediate organelle inheritance in yeast.

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance.

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

Mdm12p, a component required for mitochondrial inheritance that is conserved between budding and fission yeast.

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.