Moving Molecules into or out of Cells

Moving Molecules into or out of Cells

Small-molecule metabolic intermediates must not leak out of cells into the medium. Therefore, an impermeable membrane surrounds the cytoplasm. To solve the problem of moving essential small molecules like sugars and ions into the cell, special transporter protein molecules are inserted into the membranes. These and auxiliary proteins in the cytoplasm must possess selectivity for the small-molecules being trans-ported. If the small-molecules are being concentrated in the cell and not just passively crossing the membrane, then the proteins must also couple the consumption of metabolic energy from the cell to the active transport.

The amount of work consumed in transporting a molecule into a volume against a concentration gradient may be obtained by consider-ing the simple reaction where A_o is the concentration of the molecule outside the cell and A_i is the concentration inside the cell:

This reaction can be described by an equilibrium constant

The equilibrium constant K_eq, is related to the free energy of the reaction by the relation

∆G=RTlnK_eq

where R is about 2 cal/deg^.mole and T is 300° K (about 25° C), the temperature of many biological reactions. Suppose the energy of hy-drolysis of ATP to ADP is coupled to this reaction with a 50% efficiency. Then about 3,500 of the total of 7,000 calories available per mole of ATP hydrolyzed under physiological conditions will be available to the transport system. Consequently, the equilibrium constant will be

One interesting result of this consideration is that the work required to transport a molecule is independent of the absolute concentrations; it depends only on the ratio of the inside and outside concentrations. The transport systems of cells must recognize the type of molecule to be transported, since not all types are transported, and convey the molecule either to the inside or to the outside of the cell. Further, if the molecule is being concentrated within the cell, the system must tap an energy source for the process. Owing to the complexities of this process, it is not surprising that the details of active transport systems are far from being fully understood.

Four basic types of small-molecule transport systems have been discovered. The first of these is facilitated diffusion. Here the molecule

Figure 1.7 The cascade of reactions associated with the phosphotransferasesugar uptake system of E. coli.

must get into or out of the cell on its own, but special doors are opened for it. That is, specific carriers exist that bind to the molecule and shuttle it through the membrane. Glycerol enters most types of bacteria by this mechanism. Once within the cell the glycerol is phosphorylated and cannot diffuse back out through the membrane, nor can it exit by using the glycerol carrier protein that carried the glycerol into the cell.

A second method of concentrating molecules within cells is similar to the facilitated diffusion and phosphorylation of glycerol. The phos-photransferase system actively rather than passively carries a number of types of sugars across the cell membrane and, in the process, phos-phorylates them (Fig. 1.7). The actual energy for the transport comes from phosphoenolpyruvate. The phosphate group and part of the chemi-cal energy contained in the phosphoenolpyruvate is transferred down a series of proteins, two of which are used by all the sugars transported by this system and two of which are specific for the particular sugar being transported. The final protein is located in the membrane and is directly responsible for the transport and phosphorylation of the trans-ported sugar.

Protons are expelled from E. coli during the flow of reducing power from NADH to oxygen. The resulting concentration difference in H⁺ ions between the interior and exterior of the cell generates a proton motive force or membrane potential that can then be coupled to ATP synthesis or to the transport of molecules across the membrane.Active transport systems using this energy source are called chemiosmotic systems. In the process of permitting a proton to flow back into the cell, another small molecule can be carried into the cell, which is called symport, or carried out of the cell, which is called antiport (Fig. 1.8).

In many eukaryotic cells, a membrane potential is generated by the sodium-potassium pump. From the energy of hydrolysis of one ATP molecule, 3 Na⁺ ions are transported outside the cell and 2 K⁺ ions are transported inside. The resulting gradient in sodium ions can then be coupled to the transport of other molecules or used to transmit signals along a membrane.

Study of all transport systems has been difficult because of the necessity of working with membranes, but the chemiosmotic system has been particularly hard due to the difficulty of manipulating membrane potentials. Fortunately the existence of bacterial mutants blocked at

Figure 1.8 Coupling the excess of H⁺ions outside a cell to the transport of aspecific molecule into the cell, symport, or out of the cell, antiport, by specific proteins that couple the transport of a proton into the cell with the transport of another molecule. The ATPase generates ATP from ADP with the energy derived from permitting protons to flow back into the cell.

various steps of the transport process has permitted partial dissection of the system. We are, however, very far from completely understanding the actual mechanisms involved in chemiosmotic systems.

The binding protein systems represent another type of transport through membranes. These systems utilize proteins located in the periplasmic space that specifically bind sugars, amino acids, and ions. Apparently, these periplasmic binding proteins transfer their substrates to specific carrier molecules located in the cell membrane. The energy source for these systems is ATP or a closely related metabolite.

Transporting large molecules through the cell wall and membranes poses additional problems. Eukaryotic cells can move larger molecules through the membrane by exocytosis and endocytosis processes in which the membrane encompasses the molecule or molecules. In the case of endocytosis, the molecule can enter the cell, but it is still separated from the cytoplasm by the membrane. This membrane must be removed in order for the membrane-enclosed packet of material to be released into the cytoplasm. By an analogous process, exocytosis releases membrane-enclosed packets to the cell exterior.

Releasing phage from bacteria also poses difficult problems. Some types of filamentous phage slip through the membrane like a snake. They are encapsidated as they exit the membrane by phage proteins located in the membrane. Other types of phage must digest the cell wall to make holes large enough to exit. These phage lyse their hosts in the process of being released.

An illuminating example of endocytosis is the uptake of low density lipoprotein, a 200 Ã… diameter protein complex that carries about 1,500 molecules of cholesterol into cells. Pits coated with a receptor of the low density lipoprotein form in the membrane. The shape of these pits is guided by triskelions, an interesting structural protein consisting of three molecules of clathrin. After receptors have been in a pit for about

Figure 1.9 Endocytosis of receptor-coated pits to form coated vesicles and therecycling of receptor that inserts at random into the plasma membrane and then clusters in pits

ten minutes, the pit pinches off and diffuses through the cytoplasm (Fig. 1.9). Upon reaching the lysosome, the clatherin cage of triskelions is disassembled, cholesterol is released, and the receptors recycle.