Competition of Spontaneous Protein Folding and Mitochondrial Import Causes Dual Subcellular Location of Major Adenylate Kinase

Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form.Identifying and studying the processes and the genes that are involved in determining the rate of aging is a challenging area of modern genetics. In particular, it would be of interest to determine whether the activity of specific genes limits human life span. Several epidemiological studies of centenarians are being carried out with this goal in mind under the hypothesis that there might be a genetic basis for the exceptional life span of very long lived individuals (1). However, given the pervasive evolutionary conservation of physiological processes among organisms, a practical approach to find genes that might be involved in human aging is to first investigate the genetic basis of aging in lower organisms. The nematode genetic model system, Caenorhabditis elegans, is being extensively used to this end, and a number of genes that have been identified in this organism for their effect on aging are now also being studied in vertebrates (2, 3).The clk-2 mutants of C. elegans display a pleiotropic phenotype (reviewed in Ref. 4) that includes a slowing down of numerous physiological processes, including embryonic and postembryonic development, behavioral rates, and reproduction (5). clk-2 mutants also show an increase in life span that is particularly dramatic in combination with mutations in other genes, such as clk-1 and daf-2 (2, 6). The clk-2 mutations are temperature-sensitive (5, 7) and at 25°C produce a lethal embryonic phenotype resulting in differentiated but highly disorganized embryos (5). This is likely to be the null phenotype, since it is also produced by RNA interference at all temperatures. Extensive temperature shift experiments have demonstrated that clk-2 is required for embryonic development only during a narrow time window in which oocyte maturation, fertilization, the completion of meiosis, and the initiation of embryonic development occurs (5). Howev...

Section: Clk-2 Function Is Conserved From Yeast and Worms To Humans-mentioning

confidence: 99%

Human CLK2 Links Cell Cycle Progression, Apoptosis, and Telomere Length Regulation

Jiang

Bénard

Kébir

et al. 2003

“…S. cerevisiae survives because the broad substrate specificity of its uridylate kinase (Ura6p) compensates for the deficiency in cytoplasmic ADK activity (4). However, a small proportion of the yeast ADK (Aky2p) is localized in the mitochondrial intermembrane space (5), where it may contribute to efficient translocation of ATP from the mitochondrial matrix into the cytoplasm (1). Uridylate kinase is not present in yeast mitochondria, and hence AKY2-deficient S. cerevisiae exhibit a petite phenotype and are unable to grow under nonfermentative conditions (6).…”

mentioning

confidence: 99%

Intracellular Positioning of Isoforms Explains an Unusually Large Adenylate Kinase Gene Family in the Parasite Trypanosoma brucei

Ginger

Ngazoa

Pereira

et al. 2005

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of ␥-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates.

“…There are only a limited number of examples in which a single translation product has been shown to be distributed between two subcellular locations (1)(2)(3)(4)(5)(6). The molecular mechanisms underlying these situations have not been fully elucidated.…”

mentioning

confidence: 99%

Folding of Fumarase during Mitochondrial Import Determines its Dual Targeting in Yeast

Sass¹,

Karniely²,

Pines

2003

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Thus, fumarase processed in mitochondria returns to the cytosol. In the current work, we (i) generated mutations throughout the coding sequence which resulted in fumarases with altered conformations that are targeted to mitochondria but have lost their ability to be distributed; (ii) showed by mass spectrometry that mature cytosolic and mitochondrial fumarase isoenzymes are identical; and (iii) showed that hsp70 chaperones in the cytosol (Ssa) and mitochondria (Ssc1) can affect fumarase distribution. The results are discussed in light of our model of targeting and distribution, which suggests that rapid folding of fumarase into an import-incompetent state provides the driving force for retrograde movement of the processed protein back to the cytosol through the translocation pore.