The eukaryotic cell shows an extraordinary degree of organizational complexity. Macromolecular components that carry out different metabolic processes are segregated in distinct subcellular compartments and these must act in concert to sustain the various cellular functions. The membranes bounding all cellular organelles not only control the passage of substances between the various compartments and the surrounding cytoplasmic matrix, but also provide a framework for the functional integration and assembly of many of the organellar macromolecules into higher order complexes. This, of course, is also true for the plasma membrane, which surrounds the entire cell and regulates its interactions with the extracellular milieu.

The presence of a nucleus, a compartment limited by a membranous envelope, is the defining feature of the eukaryotic cell. In this compartment, the genome is stored and replicated and the process of decoding the genetic information begins. In the cytoplasm, several membrane-bounded organelles form an integrated endomembrane system [sometimes referred to as the "vacuolar system" (1)] which, together with the plasma membrane, is organized for the transfer of macromolecules and membrane components from one part of the cell to another, as well as to and from the cell's exterior. This transport takes place by means of membrane vesicles which bud from one organelle and fuse with another, although in some cases tubular connections may be established that carry material between organelles. The set of intercommunicating organelles that constitutes the endomembrane system (Figure 1) includes: 1) the endoplasmic reticulum (ER), which may be regarded as an extension of the nuclear envelope and serves as a major site of protein synthesis and biosynthetic activity; 2) the Golgi apparatus, which modifies many of the proteins it receives from the ER and transfers them to other sites in the cell; 3) secretory vesicles and granules, which contain proteins that have traversed the Golgi apparatus and will be released at the cell surface; 4) endosomes, which receive materials taken in from the outside of the cell within plasma membrane invaginations, and 5) lysosomes, which degrade the exogenous material from the endosome as well as endogenous cellular components. Because the luminal cavities of the several membrane-bounded compartments of the endomembrane system can communicate with each other and with the extracellular space via transport vesicles or tubular connections, all luminal faces of the membranes in this system can be regarded as topologically equivalent to each other (Figure 2).

Two other membrane-bounded organelles which do not directly communicate with the endomembrane system are found in animal cells. These are: 1) mitochondria, which generate most of the ATP required to sustain cellular activity, but also play a major role in many aspects of intermediary metabolism, and 2) peroxisomes, in which several oxidative reactions that generate hydrogen peroxide, as well as important steps in the degradation of long chain fatty acids and in the synthesis of plasmalogens and bile acids are carried out.

The portion of the cytoplasm that extends from the nuclear envelope to the plasma membrane and surrounds the membrane-bounded organelles is known as the cytoplasmic matrix (or cytomatrix). It contains filamentous elements such as microtubules, microfilaments, and intermediate filaments which constitute the cytoskeleton. This serves to organize the cytoplasm and controls the location and movement of the different organelles, and of the cell itself. The cytomatrix also contains ribosomes that function in protein synthesis, as well as numerous soluble enzymes that carry out a myriad of biochemical reactions. Several ribosomes are usually engaged in the translation of a single mRNA molecule, thus forming a polyribosome or polysome. The term cytosol is sometimes applied to the soluble components of the matrix which during cell fractionation are recovered in high speed supernatants.


Organization of Protein and Lipid Components in Membranes ( References 2, 3, 4)

Membranes are lipoprotein structures that consist of amphipathic lipids disposed in a bilayer arrangement and of proteins which penetrate the bilayer or are attached to its surfaces (Figure 3). The most abundant lipid components of membranes are phospholipids, cholesterol, and glycolipids, all of which have their polar groups facing the aqueous environment on the membrane surfaces and their hydrophobic fatty acid chains (in phospholipids and glycolipids), or the sterol ring (in cholesterol), oriented toward the membrane interior. The hydrophobic interior of cellular membranes makes them effective barriers to the passage of highly polar or charged molecules from one compartment to another. The lipid molecules within the bilayer cannot easily flip-flop from one monolayer to the other, but can undergo extensive rotational and lateral translational movements. The resulting membrane fluidity permits the lateral displacement of proteins within the plane of the membrane, which is important in membrane function.

Proteins associated with membranes fall into two categories. Those that are embedded in the phospholipid bilayer and, therefore, interact directly with the hydrophobic lipid phase are known as integral membrane proteins (Figure 3). They can only be removed from the membrane by procedures which disrupt the bilayer, such as treatment with detergents. Those proteins that do not interact directly with the membrane interior and are only bound to the surface of the membrane via interactions with other proteins or, possibly, with the polar groups of the lipids are known as peripheral membrane proteins . They can be removed from membranes by treatment with media of high ionic strength or extreme pH, or that contain chelating or chaotropic agents.

In general, the membrane-embedded portions of integral membrane proteins consist of peptide segments that are rich in hydrophobic amino acids and are approximately 20 amino acids in length, just sufficient to span the thickness of the bilayer in an alpha helical configuration. In some cases (see below) proteins are anchored in the membrane solely by a covalently bound lipid moiety. These may be the only integral membrane proteins that are exposed on only one membrane surface.

Proteins that fully traverse the lipid bilayer may cross the membrane only once and therefore have only one hydrophobic membrane-anchoring domain (Figure 4). This is the case with several well characterized hormone receptors of the plasma membrane, such as the epidermal growth factor (5, 6, see 7) and insulin receptors (8, 9, see 10), in which the ligand-binding portion of the molecule is exposed on the extracellular membrane surface while the signal-transducing domain is located in the cytoplasm. Integral membrane proteins which cross the membrane only once and have portions of their mass exposed on each surface are called bitopic proteins (11). Such proteins have one of two possible transmembrane orientations. Type I proteins (Figure 4), have their carboxy terminal ends in the cytoplasm and their amino terminal ends exposed on the extracellular surface of the plasma membrane, or on the (topologically equivalent) luminal surface of an organelle within the endomembrane system, such as the ER. Type II proteins (Figure 4) have the reverse disposition, traversing the membrane with an N, cytoplasmic, to C, extracellular or luminal, orientation. As discussed in detail below, the different transmembrane orientations of the two classes of proteins can be explained as a consequence of the mechanism by which polypeptides are inserted into the ER membrane during their synthesis.

Some transmembrane proteins, in particular ion channels, cross the phospholipid bilayer several times (Type III or polytopic proteins) (Figure 4) and the N and C terminal ends of such proteins may be found on the same or opposite sites of the membrane. The transmembrane domains of these proteins may also be hydrophobic, or may be capable of forming amphipathic helices, whose existence within the membrane may be maintained by lateral interactions with other similar helices within the same polypeptide or within other subunits of a multimeric protein. In the final configuration, a hydrophilic channel is formed by the polar faces of several helices which have their hydrophobic faces interacting with the interior of the membrane bilayer.

Many membrane proteins are glycoproteins that contain carbohydrate moieties linked to the polypeptide backbone via either N-glycosidic bonds to asparagine residues, or O-glycosidic bonds to serine or threonine residues. Carbohydrate moieties may also be linked to membrane lipids (glycolipids). In all cases, the carbohydrates of membrane components are located only on the extracellular or luminal side of the membrane . Since the enzymatic system responsible for the formation of N glycosidic bonds is present only in the ER, only proteins that reside in this organelle or pass through it during their biosynthesis can bear asparagine-linked oligosaccharide chains.


An Overview of Organelle Biogenesis (References12, 13, 14, 15)

Because of the organizational complexity of eukaryotic cells, the implementation of their genetic programs requires not only the transcription of sets of specific genes and the translation of the resulting messenger RNAs, but also the operation of mechanisms that ensure that the encoded polypeptides are transferred from their sites of synthesis to their sites of function, which may be in the cytomatrix, in a membrane, within a space enclosed by an organellar membrane, or outside the cell.

Aside from a very small number of polypeptides which are synthesized on special ribosomes found within mitochondria, the bulk of protein synthesis in mammalian cells takes place in the cytoplasmic matrix, either on ribosomes which appear to be free in the matrix, but could be associated with cytoskeletal elements, or on ribosomes which during their synthetic activity are attached to the membranes of the ER. The part of the endoplasmic reticulum to which these ribosomes are attached is called the rough ER, on account of the appearance of its cytoplasmic surface in electron micrographs. Portions of the ER devoid of attached ribosomes constitute the smooth ER.

As previously noted, the universal structural feature of all cellular membranes is the presence of a phospholipid bilayer with a hydrophobic interior that constitutes a barrier to the passage of polar molecules. In particular, proteins - which normally fold with their charged and polar residues exposed on their surfaces - cannot freely traverse a phospholipid bilayer. Therefore, special mechanisms have evolved that facilitate the incorporation of polypeptides into specific membranes, and when necessary, assist them in their passage across the hydrophobic barrier. In many cases one or more molecular chaperones (16, 17) associate with the polypeptide to be incorporated into or to be transported across the membrane and serve to maintain it in a conformation that is compatible with these processes.

Proteins destined to the nucleus, mitochondria, or peroxisomes are synthesized in ribosomes that are free in the cytoplasmic matrix, and are directly targeted to their respective organelles (Figure 5) . Specific receptors for the newly synthesized organellar proteins are present in the surface of mitochondria and probably also in the surface of peroxisomes. Those receptors must recognize structural features of each polypeptide and participate in a process that leads either to its insertion into the membrane or its translocation across it. The latter process may require the expenditure of energy and entail conformational changes or extensive structural modifications of the polypeptide. Proteins destined to the interior of the nucleus must pass through the nuclear pores of the nuclear envelope.

Some proteins of the endoplasmic reticulum and of the plasma membrane are also synthesized on free polysomes and become embedded in the membrane only after their synthesis is completed and they are discharged into the cytoplasm (Figure 5). A similar mechanism could, in principle, also lead to the insertion of proteins in the cytoplasmic surfaces of other organelles.

In contrast to the direct targeting of nuclear, mitochondrial, and peroxisomal proteins to their sites of function, proteins destined for secretion or for incorporation into lysosomes, as well as most proteins of the plasma membrane and the Golgi apparatus, are initially incorporated into the ER, and reach their sites of function by transfer through the cellular endomembrane system. Such proteins, like most proteins of the ER itself, are translocated across or inserted into the ER membrane cotranslationally, i.e., during the course of their synthesis in ribosomes bound to the rough endoplasmic reticulum membrane (Figure 6). Although these proteins may later undergo extensive posttranslational modifications, it is during or immediately after their synthesis in bound polysomes that they are either transferred to the lumen of the endomembrane system or incorporated into the membrane with a characteristic disposition with respect to the phospholipid bilayer.

After discharge into the ER lumen or incorporation into the ER membrane, proteins synthesized in membrane-bound ribosomes are subjected to sorting processes, just beginning to be understood, which ensure that certain polypeptides are retained in the ER while others are transferred to the Golgi apparatus and either remain there or, upon exit from this organelle, are transported to lysosomes, secretory vesicles or granules, or the plasma membrane. As already mentioned, transport within the endomembrane system and to and from the plasma membrane is effected by membrane vesicles that bud from one organelle, traverse a portion of the cytoplasm and fuse with another. Throughout this movement, luminal proteins remain segregated within the successive organellar cavities, and membrane proteins retain the characteristic transmembrane disposition which they acquired in the ER.

In the last few years much has been learned about the molecular machinery and intermolecular interactions involved in the formation, targetting and fusion of the vesicles that mediate interorganellar transport. This has resulted from both the development of in vitro systems that reproduce these phenomena under controlled conditions, and from the isolation of yeast mutants defective at specific stages of vesicle formation or consumption. The latter approach has led in many cases to the determination of the biochemical nature and role of the products of the defective genes.

Bibliography

(*): Reprinted with permission from "The Metabolic and Molecular Bases of Inherited Disease", Vol. I, edited by C.R. Scriver, W.S. Sly and D. Valle, The McGraw-Hilll Companies, New York, N.Y. 10020-1095 (http://www.pbg.mcgraw-hill.com).