Basis of proteins and cellular structures

As illustrated, eukaryotic cells are divided into subcellular compartments. The presence of a nuclear membrane separating the genome from the cytoplasm is the key feature which distinguishes eukaryotic ('having a true nucleus ') cells from bacteria and other prokaryotic ('pre-nuclear') cells. The nuclear membrane consists of two lipid bilayers, the inner of which is lined by a filamentous meshwork of the protein laminin. Both bilayers are traversed by nuclear pores which are cylindrical complexes of nucleoporin proteins that exhibit eight-fold symmetry, permit passive transport, and facilitate regulated passage of molecules in and out of the nucleus by interacting with a family of nuclear import and export receptors. Nuclear pores connect to short fibres extending into the cytoplasm and to longer fibres extending into the nucleus, to which molecules may 'dock' before traversing the pore. The nucleus itself is heterogeneous, with different components modulating distinct functions-for example, the nucleolus involved in generating the protein-synthesising ribosomes.

The nuclear membrane separates the nucleus from the cytoplasm. The latter contains numerous additional organelles, and filamentous biopolymers diameter from 7 nm actin filaments, through 10 nm inter ranging in mediate filaments (e.g. keratin, vimentin, desmin), to 24 nm microtubules. These cytoskeletal proteins confer rigidity upon a cell, and provide tracks by which organelles can be transported around the cell. In addition, rapid and carefully regulated polymerisation and depolymerisation of filaments are responsible for processes such as cell shape changes, pseudopodia in phagocytosis, and cellular migration.

Perhaps the best known of these are the specialised actin and myosin-based microfilaments in muscle cells which are responsible for myocyte contraction .

The ER and Golgi play critical roles in the export of secreted proteins to the extracellular environment, essentially acting as a control station to permit only mature protein to reach the cell surface. The ER is an extensive membrane-bound organelle continuous with the outer nuclear membrane,forming networks through the cytoplasm. Rough ER has associated ribosomes; ribosome-free smooth ER is particularly evident in cholesterol-synthesising cells. The Golgi apparatus consists of flattened cisternae which receive proteins from the ER and sort them for their subsequent destination. In both the ER and Golgi apparatus, extensive posttranslational modification, folding, oligomerisation and translocation of proteins occur. In addition, the ER has specific roles in lipid biosynthesis and maintenance of the ER calcium store.

Endosomes and lysosomes Endosomes transport proteins between cellular compartments and are particularly involved in the recycling of proteins internalised from the cell surface. In contrast, the acidic pH and enzymes within the similarly structured lysosomes provide a major source of protein degradation within the cell. Lysosomal storage diseases stem from inherited defects in lysosomal enzymes, resulting in the failure to degrade intracellular toxic substances.

Mitochondria are intracellular organelles essential for aerobic metabolism since they generate ATP by oxidative phosphorylation via the passage of electrons down an ionic gradient across the inner mitochondrial membrane. Although some of its structural and enzymatic components are encoded by chromosomal genes, the mitochondrial process its own small genome in which the DNA displays important differences to chromosomal DNA. In keeping with an organelle which is believed to have arisen from the symbiotica sociation of a prokaryotic bacterium, the DNA is a double stranded circular form of 16,569 bp (compared to 3 x 10 to the power 9 for the haploid genome), with 2-12 copies per mitochondrion .Other key differences to chromosomal DNA include inheritance from the maternal line (as zygotic cytoplasmic structures are derived predominantly from the oocyte). replication independent of the proliferation of the cell, and a high mutational rate due to a different DNA polymerase and lack of DNA protection by chromatin as in the nucleus. As a result, the mitochondrial DNA of somatic cells may change as the cells age, generating a heteroplasmic state with wild type and mutant DNA in the same cell. Mitochondria are most numerous in cells with high metabolic demands, as reflected in the high incidence of myopathies amongst mitochondrial disease states.

These cytoplasmic organelles contain numerous enzymes involved in the metabolism of fatty and bile acids, cholesterol, purines and amino acids. All enzymes are encoded by nuclear genes and need to be transported into peroxisomes. The inability to import proteins into peroxisomes due to perturbed peroxisome transport signals results in general peroxisomal diseases such as Zellweger's syndrome and rhizomelic dwarfism. Specific enzyme deficiencies also occur.

Protein trafficking

Proteins are transported from their site of synthesis on ribosomes to their eventual destination which may be in the cytoplasm, in subcellular compartments, membrane-bound or in the extracellular space.

Accurate protein sorting is critical not only to generate the structure of the cell and its environment, but also to enable the cell to carry out biochemical reactions efficiently.Most proteins, as they leave their site of synthesis on the large ribosomal subunit, are targeted to the rough endoplasmic reticulum by means of specific sequences at their Nterminus. In some cases, transport com commences while the polypeptide chain ide chain is still being synthesised on the ribosome. Proteins are either integrated into the ER membrane or transported through, sorted by an assembly of integral proteins. Lack of a signal sequence may result in the protein being retained in the cytoplasm by default, although there are other mechanisms to secrete proteins without a signal. For example, up-regulation of cell surface ATPase binding cassettes (which couple the hydrolysis of ATP with the secretion of such proteins) is responsible for cancer cells acquiring resistance to multiple cytotoxic drugs (via the multidrug resistance (MDR) transporter) and Plasmodium falciparum becoming resistant to chloroquine. Reduced activity of another ATPase binding cassette, the cystic fibrosis transmembrane conductance regulator (CFTR) which regulates cellular extrusion of chloride, results in cystic fibrosis.

Exchange of proteins between different subcellular compartments depends on pinching off the membrane of one compartment, vesicle formation and subsequent vesicle fusion with the target membrane. It remains a puzzle how the individual organelles manage to retai their individual compositions in the face of acquisition of vesicular contents from other compartments. Such vesicles may be 'coated with proteins derived from the cytoplasm (clathrin for endocytosed vesicles; coatamers for internal vesicles). A series of proteins on the vesicle (V-SNARES) and target membranes (t-SNAREs), together with associated soluble molecules, allow recognition of the vesicle to initiate fusion. In viral infections (of which the best studied are Haemophilus influenzae and human immunodeficiency virus-HIV), specific virus-encoded proteins undergo conformational changes to allow them to function in a fusion molecule complex and permit viral DNA entry through the lipid bilayer.

All of the information required to fold a protein correctly into its final conformation resides in the peptide sequence (primary structure) which determines the stable structure which the protein can adopt . Folding is in part determined by the nature of the peptide side-chains; for example, polar residues are directed to the exterior, nonpolar residues to the interior, and in the extracellular environment sulphur-containing side-chains form stable disulphide bonds. In addition, the flexible bonds flanking the peptide bonds direct two predictable patterns of folding of adjacent peptide chains: the a-helix,whereby a single peptide spirals around in a stable structure, critical in binding to DNA (e.g. transcription factors,) and forming the hydrophobic internal residues in membranes, and the B-pleated sheets between neighbouring peptides or opposite orientation.The combination of a-helices,B-pleated and additional from of protein secondary structure results in its final three-dimensional conformation, which currently cannot be predicted from sequence alone, but requires determination by X-ray di ince may result in diffraction studies. To increase the efficiency of such folding, the process is facilitated by additional proteins or chaperones,such as members of the heat shock protein family.

Proteins with short half-lives, or incorrectly folded proteins, are usually degraded in the cytoplasm in an ATP-mediated process, accelerated by the reversible binding of ubiquitin to lysine residues in the protein. This targets the protein to proteosomes,large protein complexes that degrate the targeted moiety into smaller peptides. Lysosomes degrate other proteins, especially those with longer half-lives.