4.2 Peptide signal sequences The distinct chemistry of proteins at the N- and C-termini provides protein molecules with two positionally and chemically unique sites for post-translational modifications and with the means to control their spatial and temporal interactions and position. This feature of proteins is crucial for a variety of biological processes from protein degradation to protein sorting for specific cellular compartments. The N- and C-termini of proteins have distinct roles, and we have already emphasised t
3.6 Membrane fusion mediated by viral proteins Until now, we have focused on the transport of material between different intracellular membrane-bound compartments and fusion of cytoplasmic membranes. This type of fusion is endoplasmic fusion. Another type of membrane fusion, called ectoplasmic fusion, is used by enveloped viruses to infect cells (enveloped viruses have an outer phospholipid bilayer). The biophysical and structural studies of viral proteins involved in the processes of membrane fusion provide a foundation for
3.4 The function of Rab proteins in directing traffic The SNARE proteins are just one component of the vesicle targeting system. Other participants in this process are the Rab family of GTPases, which regulate traffic between different cellular compartments and which are implicated in directing vesicles to their appropriate target compartments. The Rab family is the largest family of GTPases, with more than 30 members. They are distributed in specific organelles where they mediate the assembly of distinctive groups of proteins. Moreover,
3.3 Fusion of vesicles with the target membrane In this section, we shall look at how vesicles fuse with the appropriate target membrane. The targeting of different classes of transport vesicles to their distinct membrane destinations is essential in maintaining the distinct characteristics of the various eukaryotic organelles. Because coat proteins, such as clathrin, are found in different trafficking pathways, it follows that other proteins in the coat must specify the direction of transport of a particular vesicle and its ultimate desti
3.1 Introduction In the following sections, we shall describe the sequential steps involved in the movement of vesicles from one membrane to another (see Figure 9). Some of these steps are quite well defined, but for others there are gaps in our knowledge. Although we have emphasised the importance of proteins as cargo, vesicles also transfer membra
2.5 The endocytic pathways and lysosomes Endocytosis is the process by which cells internalise molecules from the outside, and it includes pinocytosis, the uptake of small soluble molecules in vesicles, and phagocytosis, the internalisation of large insoluble particles. These are two ends of a spectrum as seen microscopically, but the receptors, the subsequent intracellular trafficking pathways and the fate of the internalised molecules, vary depending on the cell type and its functions. The endocytic pathway co
2.4 Exocytosis and the secretory pathways Exocytosis is the process by which molecules are released to the outside of the cell. This includes the release of proteins to the plasma membrane and the release of secreted molecules into the extracellular fluid. All eukaryotic cells need a system to transport molecules to their plasma membrane, and many cells secrete proteins into the extracellular environment. In addition, cells in multicellular organisms communicate with each other via a variety of signalling molecules, which are
2.3 Sorting for the basolateral and apical zones of the plasma membrane Many cells are permanently polarised, and this means that surface proteins are selectively localised to different areas of the plasma membrane, depending on their function. For example, endothelial cells have adhesion molecules on the surface that contacts the basal lamina, but receptors that take up molecules from the blood (e.g. the transferrin receptor – see below) are located on the surface of the cell that is in contact with the blood. Cell surface molecules can normally diffuse latera
4.1 Glucose metabolism We are now in a position to draw together the major concepts and components of signalling, and show how they operate in one well-understood system, namely the regulation of the storage or release of glucose in the human body. From this, you will be able to recognize archetypal pathways represented in specific examples, you will be able to appreciate how the same basic pathways can be stimulated by different hormones in different tissues, and you will see how opposing hormones activate separat
3.6.2 The JAK–STAT pathway Another important protein kinase pathway is the JAK–STAT pathway. Cytokines (Section 2.2), are frequently used for signalling between cells of the immune system. Cytokine-induced signal transduction cascades are often direct pathways to the nucleus for switching on sets of genes. Janus kinases (JAKs, named after the two-faced Roman god) are a particular
6.5 Multienzyme complexes In free solution, the rate of an enzyme-catalysed reaction depends on the concentration of the enzyme and the concentration of its substrate. For an enzyme operating at suboptimal concentrations, the reaction is said to be diffusion-limited, since it depends on the random collision of the enzyme and substrate. If we consider a metabolic pathway, the product of one reaction is the substrate for the next enzyme in the pathway. Direct transfer of a metabolite from one enzyme to another wo
6.2 Catalytic mechanisms In general terms, the following mechanisms operate at the active site of an enzyme to bring about the conversion of substrate to product: Charged groups at the enzyme active site alter the distribution of electrons in the substrate. By affecting the electron distributions in key atoms in the substrate, the enzyme can destabilise existing bonds and favour the formation of new bonds. This principle is illustrated below, using as an example the hydrolysis
6.1 Introduction Among those proteins of known function, the majority are enzymes. Enzymes act as catalysts, i.e. they increase the rates of reactions, making and breaking bonds, without themselves undergoing any permanent change. They are highly specific for particular reactions and are excellent examples of how a protein's function is entirely dependent on its structure. First of all, a protein must bind its substrate (or substrates) in a specific fashion; it must then convert the substrate(s) into th
5.5 Summary of Section 5 Proteins are dynamic molecular machines. All proteins bind to other molecules, whether ions, small molecules or macromolecules, and these interactions are critical to the protein's function. The activity of proteins is regulated by changes in conformation. In allosterically regulated proteins, binding of one ligand affects the conformation of a remote part of the protein, thereby regulating interaction with a second ligand. Cooperative binding
5.3.3 Phosphorylation of proteins as a means of regulating activity Phosphorylation is an important mechanism for regulating the activity of many proteins, either switching on or switching off some activity of the protein. What protein that we have already discussed is both positively and negatively regulated by phosphorylation? Src kinase activity is switched on by dephosphorylation of 5.3.2 Cooperative binding A feature of some proteins comprising more than one subunit is that binding of a ligand to its binding site on one subunit, can increase the affinity of a neighbouring subunit for the same ligand, and hence enhance binding. The ligand-binding event on the first subunit is communicated, via conformational change, to the neighbouring subunit. This type of allosteric regulation is called cooperative binding. Haemoglobin, as we have already discussed, is a tetramer consisting of two 5.3.1 Allosteric regulation In many proteins, the binding of a particular ligand at one site affects the conformation of a second remote binding site for another ligand on the same protein. This effect is called allosteric regulation and it is an important mechanism by which a protein's binding capacity and/or its activity are regulated. Thus the switch between two different protein conformations can be controlled by binding of a regulatory ligand. 5.2 All proteins bind other molecules All proteins bind to other molecules (generically termed ligands). Ligands that can bind to proteins include: ions, e.g. Ca2+; small molecules, e.g. H2O, O2 and CO2, glucose, ATP, GTP, NAD; macromolecules, i.e. proteins, lipids, polysaccharides, nucleic acids. These interactions are specific and key to the protein's function and, of course, are critically d 4.3 Conserved protein domains By comparing the extensive protein databases, it is possible to identify many thousands of conserved domains. For example, within eukaryotes, over 600 domains have been identified with functions related to nuclear, extracellular and signalling proteins. The majority of conserved domains are evolutionarily ancient, with less than 10% being unique to vertebrates. 4.2 Amino acid sequence homologies and why they occur Consider two genes encoding proteins that have 50% of their amino acid sequence in common. How can this sequence homology be explained in terms of evolution? The most parsimonious explanation is that the similarities result from the fact that the two organisms share a common evolutionary past and that the genes encoding
