5.4Formation of Organelles and Material Transport
Selective Transport of Proteins to Organelles
Each organelle has proteins with specific functions. Synthesis of most proteins begins in ribosomes in the cytoplasm. The destination of each protein is determined by the selective signal sequence included in the amino acid sequence of the protein. This signal sequence binds to other proteins that are involved in transporting the target protein to a particular organelle. Proteins without this signal sequence remain in the cytoplasm. As shown in Figure. 5-7, selective transport has three mechanisms. The first is the transport of proteins from the cytoplasm to the nucleus through holes in the nuclear envelope (nuclear pores). The second is the transport of proteins from the cytoplasm to endoplasmic reticula, mitochondria, chloroplasts and peroxisomes, which is performed by protein translocators located in the membrane of organelles. In this case, the higher-order structure of proteins is unwound before passing through the membrane, and is folded back into its functional structure after passage. The third mechanism is transport between membrane structures, which is performed by other small membrane structures known as transport vesicles.
Transport to and from the Nucleus
The nuclear envelope that houses the DNA-containing nucleus consists of two lipid bilayers (the inner and outer membranes), and gateways known as nuclear pores cross the envelope (Fig. 5-7). Although DNA replication and DNA transcription into RNA occur in the nucleus, protein translation by ribosomes occurs in the cytoplasm. Transcribed RNA is therefore carried through the nuclear pores to the outside of the nucleus. Conversely, proteins involved in replication and transcription - such as enzymes and transcription factors - are transported into the nucleus through the nuclear pores after being synthesized in the cytoplasm.
Nernst Equation for Calculating Plasma Membrane Potential
How can the membrane potential be calculated from ion concentration? If only the K+ channel works (allowing ions to pass through the membrane) but negatively charged ions cannot pass, as shown in Figure. 5-6, the movement of K+ ions stops when the concentration gradient of K+ and the membrane potential gradient balance each other out. In such cases, the theoretical resting membrane potential can be calculated by the Nernst equation based on the ratio of ion concentration inside and outside the cell.
Assuming that only the membrane potential created by K+ (Vk) exists, the Nernst equation is expressed as follows, using Kout and Kin as the potassium ion concentration outside and inside the cell, respectively:
Vk = (RT/F) ln (Kout/Kin)
Where R is the gas constant, T is the absolute temperature and F is the Faraday constant.
In humans, assuming a body temperature of 37˚C, an extracellular ion concentration of 5.5 mM and an intracellular concentration of 150 mM, the membrane potential Vk (mV) is calculated as follows:
Vk = 62 log10 (Kout/Kin)
= 62 log10 (5.5/150)
≑ -90 mV.
Transport of Proteins to Mitochondria and Chloroplasts
Each protein synthesized in ribosomes within the cytoplasm and then transported to mitochondria or chloroplasts has a selective signal sequence. When the signal sequence of Protein A is replaced with that of Protein B in experimental genetic engineering conditions, Protein A is transported to a different organelle to which Protein B is supposed to be transported, indicating that the signal sequence determines the destination of proteins in the cell.
When proteins are transported to mitochondria or chloroplasts, their structure is unwound before passing through the outer membrane of the organelle, and is folded back into its original functional structure once inside (Fig. 5-8).
Nerve Excitation and Signaling
Neurons rapidly amplify and propagate changes in the potential of the plasma membrane as a result of ion flux. When stimuli applied to the plasma membrane temporarily open the Na+ channel, Na+ ions flow into the cell following the concentration and electric potential gradients across the membrane, thus raising its potential. This opens the Na+ channel that responds to changes in the membrane potential, which in turn causes further inflow of large amounts of Na+ ions, thus greatly raising the potential of the membrane. This phenomenon is known as action potential. The Na+ channel immediately closes, and the K+ channel that responds to changes in the membrane potential opens instead. As a result, K+ flows out of the cell following the concentration and electric potential gradients, which rapidly lowers the membrane potential. This is the mechanism of nerve impulse generation.
Such local changes in membrane potential trigger the opening and closing of nearby Na+ channels, which spreads (in one direction) the potential changes to the surrounding areas. This is the propagation of nerve excitation.
Nuclear pores are giant protein complexes with a molecular weight of over 100 million and a diameter of over 120 nm. While molecules with a weight of less than 10,000 can pass through the pores by diffusion, those with a larger molecular weight are selectively transported using the energy derived from ATP. Proteins transported to the nucleus have an amino acid sequence called the nuclear localization signal, and pass through the nuclear pores with the help of a GDP G-protein known as Ran and by bonding with importin (a transport protein). In the nucleus, Ran becomes a GTP protein, helping importin and transported proteins to dissociate from each other. The reverse reaction occurs when mRNA and proteins are transported to the outside of the nucleus.
Proteins with activity that transforms Ran to GDP proteins are abundant in the cytoplasm, and those with activity that transforms Ran to GTP proteins are abundant in the nucleus. The direction of transport into and out of the nucleus is determined in line with the difference in location of these proteins.
Proteins incorporated into the plasma membrane, enzymes in lysosomes and proteins secreted to the outside of the cell are synthesized in ribosomes attached to the endoplasmic reticulum membrane. Endoplasmic reticula with ribosomes attached are called rough endoplasmic reticula. The synthesis of a protein starts in the cytoplasm; as soon as the signal sequence of the protein is synthesized, the protein complex (SRP) recognizes and attaches itself to the sequence, and the SRP-bonded protein attaches itself to the receptor on the endoplasmic reticulum membrane, where the synthesis of the protein continues (Fig. 5-9). The protein about to be fully synthesized is transported into an endoplasmic reticulum through the protein transport channel. In the endoplasmic reticulum, the protein, with the help of proteins called chaperones, is folded into a functional form. A pair of cysteine side chains is oxidized to form a disulfide bond in the endoplasmic reticulum. In addition, many membrane proteins and proteins secreted to the outside of the cell are covalently bonded with short oligosaccharide chains.
Transport vesicles are used for the transport of membrane components and secretory proteins and for the incorporation of materials. This is known as vesicular transport (Fig. 5-10). A transport vesicle is first formed as a pit undercoated with coat proteins (Fig. 5-10) in a phenomenon known as budding. The proteins to be transported are enveloped inside the budding vesicle, which is then cut off from the endoplasmic reticulum to form a transport vesicle filled with baggage (proteins). The transport vesicle, following the detachment of the coat proteins, is carried to its destination. The membrane of the transport vesicle has a protein called SNARE, which bonds with a particular SNARE protein on the target membrane. The destination of a transport vesicle is therefore determined by the type of SNARE protein it has. Transport vesicles supply the lipids and proteins of the plasma membrane from an endoplasmic reticulum to a Goldi apparatus (Fig. 5-10A) and from a Goldi apparatus to the plasma membrane (Fig. 5-10B). When the transport vesicle is fused with the plasma membrane, proteins on the membrane stay on the cell surface, while those inside the transport vesicle are released to the outside of the cell.
Fig. 5-10. Main endoplasmic reticulum transport system
A) From an endoplasmic reticulum to a Golgi apparatus,
B) From a Golgi apparatus to the plasma membrane,
C) From the plasma membrane to an endosome,
D) From an endosome to a lysosome
Speculation on the Origin of Organelles
Organisms are divided into the categories of eukaryotes, cells with a nucleus (a membrane structure containing DNA) and prokaryotes (cells with no nucleus). Eukaryotic cells are generally larger than prokaryotic cells, feature an intracellular environment compartmentalized by membrane structures, and have a more advanced system of intracellular transport. There has been speculation regarding how organelles developed during the evolution process from prokaryotic cells to eukaryotic cells. As shown in Column Figure. 5-5, it is suggested that in ancient times, part of the plasma membrane of a prokaryotic cell with DNA and ribosomes attached was invaginated into the cell to form a nucleus enveloping DNA with two membranes. On the other hand, it has also been suggested that mitochondria and chloroplasts have evolved from different origins. Since both have a small genome that is unique to each one, these organelles are believed to have derived from different prokaryotic cells that lived symbiotically with primitive eukaryotic cells (in line with endosymbiotic theory; see Chapter 1). This speculation explains why they have two lipid bilayers (inner and outer membranes), as well as why vesicular transport, which occurs between other organelles, is not found in mitochondria and chloroplasts.
Column Fig. 5-5. Theory: mitochondria and chloroplasts originally existed as foreign cells that were engulfed by other cells
It is suggested that in ancient prokaryotic cells (1), the plasma membrane with DNA and ribosomes attached was invaginated to form a nucleus enveloped by two membranes (2), and that foreign cells were engulfed by the cell, establishing themselves as mitochondria and chloroplasts (3, 4).
Incorporation Pathways of Extracellular Materials
Extracellular materials are incorporated through the three pathways shown in Figure. 5-12. The first is, as already discussed, the direct transport of water-soluble ions and other materials to the cytoplasm by transporters or through channels in the plasma membrane.
The second pathway is called endocytosis, which involves part of the plasma membrane being incorporated into the cell in the form of transport vesicles enveloped by a coat protein called clathrin. When this occurs, proteins and lipids attached to receptors on the plasma membrane are also incorporated into the vesicles. This is also the pathway mediated by transport vesicles (Fig. 5-10C). The endosomes incorporated into the cell gradually lower their pH through the action of proton pumps, are fused with lysosomes and break down the molecules incorporated (Fig. 5-10D). Some endosome proteins are then recycled to the plasma membrane.
The third pathway is called phagocytosis, in which large particles such as bacteria are engulfed, and the plasma membrane extends toward and surrounds the target articles by the action of the cytoskeleton elements such as actin. The vacuoles formed in the cell are fused with lysosomes, leading to the degradation of ingested articles.