Mitochondria: releasing power for life and unleashing the machineries of death

The mitochondrion has long been known both as a chemical powerplant and as a cellular compartment housing various biosynthetic pathways. However, studies on the function of mitochondria in apoptotic cell death have revealed a versatility and complexity of these organelles previously unsuspected. The mechanisms proposed for mitochondrial involvement in cell death are diverse and highly controversial. In one model, mitochondria are seen as passive containers that can be made to leak out cytotoxic proteins. In other scenarios, however, certain more or less familiar aspects of mitochondrial physiology, such as oxidative phosphorylation, generation of oxygen radicals, dynamic morphological rearrangements, calcium overload, and permeability transition, are proposed to play crucial roles. In this review, we examine a few promising mechanisms that have been gaining attention recently.     

A role for subunit III in proton uptake into the D pathway and a possible proton exit pathway in Rhodobacter sphaeroides cytochrome c oxidase

Protons are transferred from the inner surface of cytochrome c oxidase to the active site by the D and K pathways, as well as from the D pathway to the outer surface by a largely undefined proton exit route. Alteration of the initial proton acceptor of the D pathway, D132, to alanine has previously been shown to greatly inhibit oxidase turnover and slow proton uptake into the D pathway. Here it is shown that the removal of subunit III restores a substantial rate of O(2) reduction to D132A. Presumably an alternative proton acceptor for the D pathway becomes active in the absence of subunit III and D132. Thus, in the absence of subunit III cytochrome oxidase shows greater flexibility in terms of proton entry into the D pathway. In the presence of DeltaPsi and DeltapH, turnover of the wild-type oxidase or D132A is slower in the absence of subunit III. Comparison of the turnover rates of subunit III-depleted wild-type oxidase to those of the zinc-inhibited wild-type oxidase containing subunit III, both reconstituted into vesicles, leads to the hypothesis that the absence of subunit III inhibits the ability of the normal proton exit pathway to take up protons from the outside in the presence of DeltaPsi and DeltapH. Thus, subunit III appears to affect the transfer of protons from both the inner and outer surfaces of cytochrome oxidase, perhaps accounting for the long-observed lower efficiency of proton pumping by the subunit III-depleted oxidase.     

Molecular identification of high and low affinity receptors for nicotinic acid

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.     

A Novel Membrane Protein Is a Mouse Mammary Tumor Virus Receptor

Mouse mammary tumor virus (MMTV) infects a number of different cell types, including mammary gland and lymphoid cells, in vivo. To identify the cellular receptor for this virus, a mouse cDNA expression library was transfected into Cos-7 monkey kidney cells, and those transfected cells able to bind virus were selected by using antibody against the virus’s cell surface envelope protein, gp52. One clone isolated from a library prepared from newborn thymus RNA, called MTVR, was able to confer virus binding to both monkey and human cells; this binding was blocked by anti-MTVR antibody. Moreover, transfection of MTVR into CV1 cells rendered them susceptible to infection by a murine leukemia virus-based retrovirus vector pseudotyped with the MMTV envelope protein. An epitope-tagged MTVR cofractionated with cellular membranes. Coimmunoprecipitation of the MMTV envelope protein and a MTVR-rabbit Fc fusion protein showed that these two proteins bound to each other. The MTVR sequence clone is unique, shows no homology to known membrane proteins, and is transcribed in many tissues.     

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.     

Protein phosphorylation during the asexual life cycle of the human malarial parasite Plasmodium falciparum

Recent evidence has suggested that extensive changes in the phosphorylation profile of red cell membrane proteins are associated with the invasion and development of the malarial parasite. In order to further define the role of parasite protein phosphorylation in these events we have looked at this phosphorylation using: (1) continuous metabolic labelling with [32P]orthophosphate, (2) pulse-labelling with [32P]orthophosphate and [35S]methionine, (3) autophosphorylation of infected cells using [gamma-32P]ATP, (4) invasion of prelabelled red cells. Many parasite proteins were labelled, some differentially according to the phosphorylation protocol employed, and we were able to partially characterise several of the major parasite phosphoproteins in terms of their association with host cell membrane and the stage specificity of phosphorylation.     

Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1–Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.     

Limnology of two lake systems of Katmai National Park and Preserve, Alaska: Part II. Light penetration and Secchi depth

Seven large lakes in the Naknek River drainage and four in the Alagnak River drainage within the Katmai National Park and Preserve, Alaska, were surveyed once a summer during the period 1990–92 to determine baseline limnological conditions. All of the lakes are oligotrophic based on Secchi depth (SD) transparency and light penetration. Overall, SD transparency varied from 4.4 m to 17 m, the vertical light extinction coefficient (K _d) ranged from 0.411 m^-1 to 0.070 m^-1 and the depth of 1% light penetration (I_1%) varied from 11 m to 67 m. However, because of greater light scattering, the percent of photosynthetic radiation (PAR) at SD was nearly twice as much in Battle Lake (30.4%) and Naknek Lake (32.8%), compared with the other nine lakes (mean 16%). Consequently, the ratio of I_1% to SD was about 4 in these two lakes compared to a mean value of 2.6 for the other lakes. However, Battle Lake is a deep blue calcium sulfate lake with little phytoplankton, whereas Naknek Lake contains some inorganic glacial flour and volcanic ash, as well as planktonic algae, but where sampled exhibits minimal turbidity. Biomass of planktonic algae (indexed by total chlorophyll concentration) explained most of the variation in SD (r ²=0.66), K _d (r ²=0.75), and I _1% (r ²=0.85). In contrast, neither color nor turbidity were significant predictors of any optical variable. Considering all 11 lakes, there was a significant linear relationship between SD and both K _d (r ²=0.80) and I _1% (r ²=0.72); however, most of the unaccounted for variation was attributed to Battle Lake and Naknek Lake. Although changes in water transparency are often linked to changes in algal biomass (chlorophyll), simple measures of SD transparency alone may not be appropriate for assessing whole-scale watershed or regional changes toward oligotrophication or eutrophication in lakes of the remote and pristine Katmai National Park and Preserve.