Experientia最新文献

A key statement of the 1965 Monod-Wyman-Changeux (MWC) model for allosteric proteins concerns the distinction between the ligand-binding function (Y) and the relevant state function (R). Sequential models predict overlapping behavior of the two functions. In contrast, a straightforward experimental consequence of the MWC model is that for an oligomeric protein the parameters which characterize the two functions should differ significantly. Two situations, where R > Y and the system is hyper-responsive or where R < Y and the system is hypo-responsive, have been encountered. Indeed, the hyper-responsive pattern was first observed for the enzyme aspartate transcarbamoylase, by comparing Y with R monitored by a change in sedimentation. Extensions of the theory to ligand-gated channels led to the suggestion that, on the one hand, hyper-responsive properties also occur with high-affinity mutants. On the other hand, native channels of the acetylcholine neuronal alpha 7 receptor and low-affinity mutants of the glycine receptor can be interpreted in terms of the hypo-responsive pattern. For the ligand-gated channels, whereas R is detected directly by ion flux, ligand binding has rarely been measured and the formation of desensitized states may complicate the analysis. However, stochastic models incorporating both binding and channel opening for single molecules predict differences that should be measurable with new experimental approaches, particularly fluorescence correlation spectroscopy.

Our level of understanding of peroxisome biogenesis in comparison with other cellular organelles is rudimentary, yet the fragments of information available indicate that the targeting and import of peroxisomal proteins occur by fundamentally different mechanisms. Genetic studies have identified a number of genes required for peroxisome assembly, but in most cases the functions of the gene products remain unknown. In vitro protein translocation systems have played a prominent role in unravelling the biochemistry of protein translocation into other organelles. This review considers some of the requirements for establishing a bona fide peroxisomal import assay and discusses the findings which have emerged as a result of using such experimental systems.

Targeting signals of mitochondrial precursors are cleaved in the matrix during or after import by the mitochondrial processing peptidase (MPP). This enzyme consists of two nonidentical alpha- and beta-subunits each of molecular weight of about 50 kDa. In mammals and fungi, MPP is soluble in the matrix, whereas in plants the enzyme is part of the cytochrome bc1 complex. MPP is a metalloendopeptidase which has been classified as a member of the pitrilysin family on the basis of the HXXEHX76E zinc-binding motif present in beta-MPP. Both subunits of MPP are required for processing activity. The alpha-subunit of MPP, which probably recognizes a three-dimensional motif adopted by the presequence, presents the presequence to beta-MPP, which carries the catalytic active site. MPP acts as an endoprotease on chemically synthesized peptides corresponding to mitochondrial presequences. Matrix-targeting signals and MPP cleavage signals seem to be distinct, although the two signals may overlap within a given presequence. The structural element helix-turn-helix, that cleavable presequences adopt in a membrane mimetic environment, may be required for processing but is not sufficient for proteolysis. Binding of the presequence by alpha-MPP tolerates a high degree of mutations of the presequence. alpha-MPP may present a degenerated cleavage site motif to beta-MPP in an accessible conformation for processing. The conformation of mitochondrial presequences bound to MPP remains largely unknown.

Vacuolar H(+)-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V1) and membrane (V0) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPase that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by 'drinking' the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.

Mitochondrial gene expression in yeast, Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These unclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.

The Ca2+ pump of the plasma membrane (PMCA) is regulated by a number of agents. The most important is calmodulin (CaM), which binds to a domain located in the C-terminal portion of the pump, removing it from an autoinhibitory site next to the active site. The CaM-binding domain is preceded by an acidic sequence which contains a hidden signal for endoplasmic reticulum (ER) retention. Chimeras of the PMCA and endoplasmic reticulum (SERCA) pumps have revealed the presence of a strong signal for ER retention in the first 45 residues of the SERCA pump. Four gene products of the PMCA pump are known: two of them (1 and 4) are ubiquitously expressed, two (2 and 3) are specific for nerve cells and may be induced by their activation. Mutagenesis work has identified four residues in three of the transmembrane domains of the pump which may be components of the trans-protein Ca2+ path. The mutation of two of these residues alters the membrane targeting of the pump.

Endocytosis is a general term that is used to describe the internalization of external and plasma membrane molecules into the cell interior. In fact, several different mechanisms exist for the internalization step of this process. In this review we emphasize the work on the actin-dependent pathways, in particular in the yeast Saccharomyces cerevisiae, because several components of the molecular machinery are identified. In this yeast, the analysis of endocytosis in various mutants reveals a requirement for actin, calmodulin, a type I myosin, as well as a number of other proteins that affect actin dynamics. Some of these proteins have homology to proteins in animal cells that are believed to be involved in endocytosis. In addition, the demonstration that ubiquitination of some cell surface molecules is required for their efficient internalization is described. We compare the actin, myosin and ubiquitin requirements for endocytosis with recent results found studying these processes using Dictyostelium discoideum and animal cells.

Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. In Saccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of the Escherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK and S. cerevisiae Ssa1p, respectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of approximately 70 residue similarity to the 'J box', the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.

Peroxisomes are vital intracellular organelles which house enzymes involved in a variety of metabolic pathways. The large number of human disorders associated with flawed peroxisome biogenesis emphasizes the importance of protein targeting to, and translocation across, the peroxisomal membrane. This brief review will summarize some of the emerging themes of peroxisomal protein import, specifically addressing the targeting signals possessed by constituent proteins, as well as the cytosolic, membrane and luminal components of the import machinery. Although a detailed understanding of the molecular mechanisms of peroxisomal protein import is not yet available, remarkable progress has been made in the field in recent years. An overview of these advances will be presented.