CELL STRUCTURE AND FUNCTION 8, 91-107 (1983) C by Japan Society for

Subcellular Compartments and Topogenesis

Yutaka Tashiro

Department of Physiology, Kansai Medical University, Osaka 570, Japan

ABSTRACT. A cell is surrounded by a plasma membrane. It contains vari- ous , most of which are enclosed by limiting membranes. The intracellular space is thus divided into a number of subcellular compartments. Structurally, a cell is composed of membranes and the spaces enclosed by those membranes. In order to classify these compartments, the extracellular space has been designated Si and whenever a unit membrane structure is crossed to arrive at the next space, one is added to term; the cytoplasmic space becomes S2,the intraluminal space of the and the intermembrane space of the mitochondria S3, and the matrix space of the mitochondria S4. Similary, the plasma membrane is M1 , the outer membrane of the mito- chondria M2, and the inner counterpart M3. This classification of the subcellular compartments is useful in under- standing a number of complicated cellular structures and functions. The intracellular transport of newly synthesized protein (protein topogenesis) and the probable development of subcellular organelles during phylogenesis of eukaryotic cells is discussed in terms of these subcellular compartments.

I. Introduction

A cell is a markedly heterogeneous system surrounded by plasma membranes. It contains various bodies called cell organelles; e.g., the nucleus, mitochondria and endoplasmic reticulum (ER). Most organelles are enclosed by the limiting membranes ; thus, the intracellular space is divided into a number of subcellular compartments that presumably are endowed with particular microenvironments. The efficient performance of extremely complicated and diverse cellular functions in a very limited space, only 200-1,500 ƒÊm3, may depend largely on the existence of a number of these complicated subcellular compartments. All cellular functions ; intracellular transport of newly synthesized (sub- cellular protein topogenesis), protein catabolism, maintainance of particular intracellular ionic environments and metabolic pools etc., appear to be intricately correlated with subcellular compartmentalization. For example, the route for the intracellular transport of secretory proteins was postulated by Palade as always in the luminal spaces of the ER, and secretory vesicles (15, 22, 40). This has been confirmed by a number of studies and the importance of the segregation of secretory proteins in these luminal spaces is well recognized (4, 49). The involvement of lytic compartments in intracellular catabolic processes has been stressed by de Duve since the discovery of (11). The segregation of calcium ion in the luminal space of the sarcoplasmic reticulum and this ion's roles in

91 92 Y. Tashiro the regulation of muscle contraction has been described by Ebashi (13). Compart- mentation of respiratory and the vectorial transport of protons in the cristal membranes of mitochondria is essential in Mitchell's chemiosmotic hypothesis of ATP formation (35). Some of these problems have been discussed in a symposium on Microenvironments and Metabolic Compartmentation (53). In this review, the definition of subcellular compartments is given, then the sub- cellular compartments of prokaryotic and eukaryotkc cells are compared. As an example, protein topogenesis is explained in terms of subcellular compartments. The probable development of subcellular compartments during phylogenesis in eukaryotic cells also is discussed.

II. Nomenclature

Structurally, the cell is composed of two kinds of compartments; the membrane (M) and the space (S) enclosed by a membrane. The former includes both the plasma membrane and the limiting membranes of the various cell organelles. When body fluid compartments are considered in physiology, the boundaries between them usually are neglected. For example, the cytoplasmic membrane, which separates the intracellular and extracellular fluids, is assumed to be simply a spaceless boundary between the two compartments. When we consider subcellular compartments, however, the membrane spaces can not be neglected. First, the volume occupied by the membrane space is not negligible in such tiny structures as cell organelles. If there is a spherical cytoplasmic vesicle 100 nm in diameter with a 10 nm width of limiting membrane, the ratio for the volumes of the membrane and the intraluminal space is almost 1 : 1. Although this ratio decreases to 1 : 16 when the diameter of a vesicle is 1,000 nm, the membrane space can not be neglected because of the extremely large area made up of the total membrane surfaces in a cell. The average total membrane surface area per gram of liver has been estimated as 9.3 m2 by Weibel et al. (7). In addition, all the biomembranes are composed of lipids and proteins, and they

Fig. 1. Designations of the various cellular compartments. See the text for their explanation. Subcellular Compartments 93 always contain double layers of lipids. Thus the membrane structures provide the cell hydrophobic domains and amphiphilic surfaces, in and on which a number of hydrophilic, amphiphilic and hydrophobic substances can interact efficiently. As there are a number of complicated compartments within the cell, I will designate the extracellular space Si and add one whenever a unit membrane structure is crossed to arrive at the next space (Fig. 1). Thus the cytoplasmic space is S2 and the intraluminal spaces of cytoplasmic organelles such as the ER and Golgi apparatus is S3. The surface of a membrane is not always smooth; complicated structures such as invagination and infolding are occasionally observed. These spaces may have a micro- environment which differs from the environment of the bulk space. Such a space is designated by a symbol such as S1'. If more infolding occurs on the infolded membrane structure, this space is indicated by a double prime like Si". Similary, plasma membrane is M 1, and the next membrane within the cytoplasmic space (S2) is M2, and so on. As the infolded membrane may differ from the bulk membrane from which the infolding was made, it is shown by prime symbol as is M2'. There are various kinds of cell organelles within the . To discriminate between the space and the membrane of the , the name of each organelle is

TABLE 1. 94 Y. Tashiro added by the two letter code shown in Table 1. When it is necessary to further dis- criminate between substructures within each organella, a three letter code is used.

III. Subcellular compartments in prokaryotic cells Compartmentation in bacteria is shown in Fig. 2. The outer surface of a bacterium is surrounded by a which is composed mainly of peptidoglycan and other components specific for gram-positive or gram-negative cell walls (23). The cell wall of a bacterium is not a membrane as defined here. The aqueous compartment between the cell wall and cytoplasmic membrane is the periplasmic space which contains the various binding proteins required for nutrient transport and such hydrolytic enzymes as phosphatase, nuclease, protease and penicillinase (2). The microenvironment in the periplasmic space definitely differs from that of the bulk outer space (S1); therefore, the periplasmic space is S1'. Sometimes deep infoldings in the cytoplasmic membrane, mesosomes, are present. These form a new subcompartment, S1". In some bacteria, invagination (S1") and formation of cytoplasmic vesicles (S3) are present, particulary, in photosynthetic bacteria. In the cytoplasmic space (S2), a number of are attached to the cyto- plasmic membrane.

Fig. 2. Compartments of a prokaryotic cell. In Figs. 2-7, the space compartments were given as numbers without the S. Ml, M2, etc. are the membrane compartments. The replication origin of DNA was assumed to be attached to the cytoplasmic membrane as suggested by Jacob et al. (21). Closed circles indicate ribosomes.

IV. Subcellular compartments in eukaryotic cells Compartmentation in eukaryotic cells is much more complicated than in prokaryo- tic cells (Fig. 3). The nomenclature has been explained above (II). The various sub- cellular compartments can be summarized as follows : A. Membranes (M) M1 : Cytoplasmic membrane (CM) M2: All limiting membranes of cell organelles. M3: Inner membranes of mitochondria and . Intraluminal vesicles in the cytoplasmic organelles such as multivesicular bodies. M4: Thylakoid membrane of the . Isolated mitochondrial cristae hich are not connected to the inner mitochondrial membrane. Subcellular Compartments 95

Fig. 3. Compartments of a eukaryotic cell. Abbreviations for the intracellular organelles shown in Figs. 3-7 are given in Table 1.

B. Spaces (S) S1 : Extracellular space S1' : Extracellular space surrounded by invaginated or infolded cytoplasmic membrane. Extracellular space in coated pits or caveoles. S2: Cytoplasmic space. S2' : Nuclear matrix space. S3: Luminal spaces of various cytoplasmic organelles. Intermembrane spaces of mitochondria and chloroplasts. S3' : Intracristal space of mitochondria. S4: Mitochondrial matrix and chloroplast stroma space. Luminal space in small vesicles in the multivesicular bodies. S5: Intrathylakoidal space in the chloroplast. Intracristal space in isolated mitochondrial cristae.

Structurally, the membraneous cell organelles are covered by a single membrane or by a double membrane (envelope). The nucleus, mitochondria and chloroplasts belong to the latter ; all contain DNA. Compartmentalization of the nucleus, however, differs greatly from that of the mitochondria and chloroplasts. The nuclear matrix space is connected directly to the cytoplasmic space by a number of nuclear pores, through which macromolecules such as mRNA, ribosomes and histones as well as small ions pass easily. The nuclear matrix space is, therefore, not S3 but S2'. The outer nuclear membrane of the nuclear envelope occasionally is continuous 96 Y. Tashiro with the rough ER membrane (31) and is very similar to it in a number of properties. The inner nuclear membrane, however, appears to differ greatly from its outer counterpart; the latter does not contain cytochrome P-450 (30). Mitochondria are covered by outer and inner mitochondrial membranes. The space between the two is the intermembrane space (S3). The outer mitochondrial membrane contains porin which forms channels for the nonspecific passage of low molecular weight hydrophilic solutes (56). This microenvironment of the inter- membrane space may be markedly modified by the appearance of porin in the outer membrane (legend to Fig. 7). Although this mitochondrial porin is functionally similar to the bacterial porin on the outer membrane of Gram-negative bacteria, the outer mitochondrial membrane is assumed to be a true membrane (as defined here), and is radically different from the bacterial cell wall. The inner mitochondrial membrane is infolded deep into the matrix space (S4-MT) as mitochondrial cristae. The space surrounded by the cristal membrane is the intracristal space, S3', because it usually is connected to the intermembrane space. If it is not, the isolated intracristal space is S5. Although the continuity of the intra- cristal space with the intermembrane space of the has been established by Palade (38, 39), the possibility remains that this continuity is occasionally lost, even for a short time. Compartmentalization in the chloroplast is similar to that in the mitochondrion. The intrathylakoidal space (S5), however, is not continuous with the intermembrane space (S3), as in the intracristal space of the mitochondrion and always functions as an independent compartment. Compartmentalization of the other membraneous organelles is very simple. They usually contain only an intraluminal space (S3). Occasionally, however, small vesicles exist within the intraluminal space as in the multivesicular bodies. The intraluminal space of such vesicles is S4.

V. Relationships between subcellular compartments

Subcellular compartments in prokaryotic and eukaryotic cells have been defined in the previous sections. A comparison of the various compartments shows that there are some rules in the relationships between subcellular compartments. 1. Spaces with even or odd number have similarities with members of their own subgroup, but not with others. That is, the spaces with even number are always cellular matrix space. The spaces with odd number are extramatrix space which could be connected to the extracellular space under certain conditions. (1) (2) 2. Each membrane compartment is different greatly. Thus, (3) Even the membranes with the same number usually differ. For example, (4) The existence of marker enzymes specific for each membrane is a clear indication of the specificity of each membrane compartment. Some examples of these rules are explained in the following sections. Subcellular Compartments 97

VI. Subcellular compartments and topogenesis of proteins newly synthesized on free ribosomes Almost all the cellular functions may be intimately correlated with subcellular compartmentalization. As an example, the intracellular transport of proteins (protein topogenesis) will be considered in terms of subcellular compartments. Some general reviews of protein topogenesis have been published recently (3, 50). All the cytoplasmic ribosomes are located in the cytoplasmic space (S2) ; therefore, all protein synthesis takes place in S2 except for protein synthesis in mitochondria which takes place in the matrix space (S4-MT) and that in the chloroplasts which take place in the stroma space (S4-CP). There are two kinds of ribosomes ; free ribosomes, and membrane-bound ribosomes which are attached to the cytoplasmic surface of ER membranes. Siekevitz and Palade (54) found that secretory proteins are synthesized on membrane bound ribosomes. Since then it was revealed by a number of investigators that free and membrane bound ribosomes synthesize different classes of proteins (45). At least five classes of protein are synthesized on free ribosomes (Fig. 4). 1. All the soluble and insoluble cytosolic proteins including the contractile and cytoskeletal proteins. 2. All the nuclear proteins in S2'-NU, such as histones and nuclear acidic proteins. 3. Some membrane proteins on the cytoplasmic face of the organelles and plasma membranes. 4. Peroxisomal proteins

Fig. 4. Routes of intracellular transport of proteins synthesized on free ribosomes in the cyto- plasmic space (S2). 98 Y. Tashiro

5. Most mitochondrial and chloroplast proteins of the outer (M2), inner (M3), cristal (M3') and thylakoidal membranes (M4) and those in the intermembrane (S3-MT or S3-CP), intracristal (S3'- or S4-MT) and intrathyrakoidal (S4-CP) spaces. In the case of cytosolic proteins, the site of synthesis and the site of localization are in the same compartment, S2. There appears to be no difficulty for the topogenesis of this kind of proteins, except in extreme cases ; in axonal transport in neurons, free cytosolic proteins (e.g. clathrin) have to travel long distances from the site of synthesis (soma) to the site of localization in the synaptic knobs. Most nuclear proteins probably are transported through the nuclear pores. The molecular mechanisms for this transport have barely begun to be investigated. Recently a special polypeptide domain has been reported that specifies the migration of nucleoplasmin into the nucleus (12). Some membrane proteins (integral and peripheral) found in, or on, the cytoplasmic faces of various organelles, such as cytochrome b5 and cytochrome b5 reductase, are synthesized on free ribosomes, then post-translationally incorporated into the cytoplasmic faces of the membranes (8, 37, 41). The myelin basic protein in glia cells, which is synthesized on free ribosomes and incorporated onto the cytoplasmic face of the cytoplasmic membrane during myelination is another such protein (9). It is postulated that these membrane proteins are post-translationally bound to and inserted into the cytoplasmic surface of the organellar membranes by using a hydro- phobic sequence at their C-termini (50). Peroxisomal enzymes such as catalase and uricase have been reported to be synthe- sized on free ribosomes and to be post-translationally transported into the luminal cavity of the (43, 18). No difference in molecular weight was detected between the nascent and authentic enzymes. Some mitochondrial and chloroplast proteins are synthesized by their own ribosomes. But most are synthesized on cytoplasmic-free ribosomes in S2 and post-translationally transported into the mitochondria. These proteins include the mitochondrial proteins found in almost all the compartments in the mitochondria and chloroplasts (10). They are usually synthesized as precursor proteins that are 2,000-4,000 daltons larger than the corresponding authentic proteins. The excess polypeptide chains are cleaved when the precursor proteins are incorporated into the organelles then inserted into the destined sites. Topogenesis of cytochrome c to the luminal side of inner mitochondrial membrane is an exceptional case in which only N-terminal methionine is cleaved then glycine is acetylated in S2 and transported into the mitochondria and inserted into the luminal face of the inner mitochondrial membrane, probably by on addressing signal in the C-terminal region of the molecules (29). In the case of chloroplast proteins, the small subunit of ribulose-1,5-bisphosphate carboxylase has been studied extensively by Chua and his co-workers (10). They report that it is synthesized on free cytoplasmic ribosomes as a precursor protein 4,000 daltons larger than the authentic protein, and that the transit sequence located at the N-terminus is cleaved post-translationally during topogenesis of the small subunit protein. Subcellular Compartments 99

VII. Subcellular compartments and topogenesis of proteins newly synthesized on membrane-bound ribosomes

What kinds of protein are synthesized on the membrane bound ribosomes? As stated above, Siekevitz and Palade (54) have shown that secretory proteins are synthesized on membrane-bound ribosomes. Because secretory proteins are to be secreted to the extracellular space (S1) and ribosomes are present in S2, these

proteins must cross the membrane at least once. Palade (40) suggested that the intracellular route of transport for secretory proteins is S2•¨ S3-ER•¨ S3-TV•¨ S3-GO •¨ 53-SV S1. The first step (S2•¨ S3-ER) includes transmembrane transport of newly synthesized proteins from S2 to S3. The question is where and how this transport take place. It has been shown that ribosomes are attached to the ER membrane through their large subparticles (60 S), and secretory proteins synthesized on membrane bound ribosomes are cotranslationally and vectorially transported through a hypothetical channel in these large subparticles and the ER membrane to the intracisternal cavity (42, 48, 51). Subsequently signal hypothesis has been proposed to explain how polysomes that synthesize secretory proteins become membrane bound (5, 6). Signal sequences have been found on all the N-termini of the secretory proteins (25), except ovalbumin in which the non-cleavable insertion sequence has been identified within the molecule

(26).

Fig. 5. Routes of intracellular transport of proteins synthesized on ribosomes attached to the endoplasmic reticulum. Route 1: secretory proteins; route 2: lysosomal proteins. Membrane proteins probably are transported along with these proteins. A possible route for the recycling of plasma membrane proteins by endocytosis is indicated. 100 Y. Tashiro

The discovery of the signal recognition protein (56) and its receptors on the ER membrane (17) has greatly advanced our understanding of the molecular control mechanism involved in this transmembrane transport process. Once secretory proteins are transported to the intracisternal cavity of the ER

(S3-ER), they are transported intracellularly via S3 of various organelles as suggested by Palade (40) and as shown in Fig. 5; from the intracisternal space of the ER to the Golgi apparatus (S3-GO) via transfer vesicles (S3-TV), then to secretory vesicles

(S3-SV). The final step in the secretory process is exocytosis. This process involves fusion of the membrane of the secretory vesicles with the plasma membranes which releases secretory proteins into the extracellular space (S3•¨ S1). This process is not trans- membrane transport; it is simply translocation of secretory proteins from S3 to S1 which are similar (S3 •¬S1). Reports have been made (14, 40, 46) that, in addition to secretory proteins, lyso- somal proteins are synthesized on membrane-bound ribosomes as precursor proteins then transported vectorially into the intracisternal space of the rough ER (S2 •¨S3- RER), then to the Golgi apparatus (S3-GO). At the trans region of the Golgi appara- tus, lysosomal proteins may be packed into small vesicles. The primary lysosomes

(S3-PLY) thus formed would be stored in the trans Golgi region, ready to fuse with heterophagosomes (HPH) or autophagosomes (APH) to become secondary lyso- somes (SLY). It has been suggested that the post-translational modification of lysosomal hydrolase precursors by the addition of phosphomannosyl residues to the oligo- saccharide chain is necessary to divert the lysosomal enzymes from the secretory pathway and direct them to their destination (24). Most secretory proteins, and probably all of the lysosomal proteins, are glyco- proteins. All the processes of glycosylation are carried out in S3 ; the initial steps in the assembly and processing of N-linked oligosaccharide chains in S3-RER with further processing and terminal glycosylation in S3-GO, where the synthesis of -linked oligosaccharide chains is also carried out (19). S3-ER, S3-GO, S3-SV, O

S3-LY, etc. are thus the intracellular compartments which are rich in the carbo- hydrate chains of glycoproteins and glycolipids.

VIII. Subcellular compartments and topogenesis of membrane proteins The third group of proteins synthesized on membrane-bound ribosomes are membrane proteins. In fact, almost all cellular membrane proteins are synthesized there with the exceptions of mitochondria' and chloroplast proteins (IV-5) and some cytoplasmic face membrane proteins that are synthesized on free ribosomes (IV-2). According to the relation of the phospholipid bilayer and the type of interaction with other membrane components, five kinds of (MP) have been classified (49, 52): luminal side peripheral and integral membrane protein (PMP, IMP), transmembrane (integral membrane) protein (TM-IMP or TMP) and cyto- plasmic side PM P and IMP. The schematic dispositions of these membrane proteins during intracellular transport and in the plasma membrane are shown in Fig. 6. The topogenesis of transmembrane proteins and luminal side membrane protein have been described in detail by Blobel (3), Lodish et al. (27) and Sabatini et al. (50). The transport mechanism for these proteins is similar to that for secretory proteins, Subcellular Compartments 101

Fig. 6. The biosynthesis, intracellular transport and final disposition of membrane proteins. Monotopic cytoplasmic side ER membrane proteins are synthesized either on membrane-bound ribosomes ;e.g., cytochrome P-450) or on free ribosomes (e.g., cytochrome b5) and inserted directly into the cytoplasmic face of ER membrane. The membrane glycoproteins, which are destined for plasma membrane, are synthesized on the rough ER, transported to and processed in the Golgi apparatus and finally inserted into the plasma membrane. The forked figure denotes an asparagine- linked oligosaccharide. Possible disposition of bitopic N-C type integral membrane protein (e.g., vesicular stomatitis virus G protein and human histocompatibility antigen (HLA-A) ), biotopic C-N type integral membrane protein (e.g., sucrose-isomultase), polytopic integral membrane protein; e.g., the erythrocyte band 3 protein) are from a review by Lodish et al. ; 27). The dotted lines indicate the peripheral polypeptides which are attached to intergral proteins by secondary forces. but the membrane proteins have a stop-transfer sequence by which they are firmly anchored to the phospholipid bilayer of the ER membrane. When the insertion signal sequence of the membrane proteins is located at the N-terminus and is cleavable (as in the case of secretory proteins) and the stop transfer sequence is located at the C-terminal region, bitopic N-C type (orthodox type) membrane protein is formed usually leaving a short cytoplasmic segment composed of 30 amino acid residues on the cytoplasmic side of the ER membrane. When the insertion signal sequence at the N-terminus is non-cleavable, the N- terminal region of the membrane protein remains on the cytoplasmic side and the C-terminal region is transferred across the ER membrane to the luminal side, forming 102 Y. Tashiro a bitopic C-N type (reverse type) membrane protein. When there are more than one insertion signal and stop-transfer sequences within a single membrane protein, a polytopic transmembrane protein is formed which traverses the phospholipid bilayer more than once, for example in the biosynthesis of the band 3 protein of erythrocytes (50). How are the cytoplasmic side membrane proteins incorporated into the ER membrane? We showed that NADPH-cytochrome reductase (36) and cytochrome P-450 (16) are inserted directly from membrane bound ribosomes into the ER mem- brane. These proteins have noncleavable hydrophobic domains at the N-terminal region (1, 21) by which the proteins presumably are inserted contranslationally to the cytoplasmic side of ER membranes (16, 36). We have shown that ferritin antibody conjugates againts cytochrome P-450 do not bind to the luminal surface of the outer nuclear envelope (30). This means that cytochrome P-450 is probably not a trans- membrane protein, but is a monotopic cytoplasmic face integral membrane protein (Fig. 6). As described above, all membrane proteins except those for the mitochondria and chloroplasts are synthesized exclusively on the rough ER. This means that membrane proteins destined for cytoplasmic organelles other than the ER are transported from the ER to the specific organelles Golgi apparatus, secretory vesicles, lysosomes and plasma membranes, etc. Each organella is reported to have its own characteristic membrane proteins or enzymes (marker proteins or marker enzymes), which means that membrane proteins are sorted out during their transport processes. For example, the protein composition of the Golgi membrane differs from that of the ER (34). Two possibilities have been proposed to explain the sorting process of the mem- brane proteins in the transport from ER to Golgi apparatus. Palade (1) suggested that the membrane proteins of both the ER and Golgi membranes are transported via transfer vesicles from the ER to the innermost cisternae of the Golgi apparatus (forming face), then only the ER proteins are collected (presumably at the lateral dilated rims of the cisternae) to form transfer vesicles, after which they are sent back to the ER by these vesicles (shuttle mechanism). The distillator model of the Golgi apparatus proposed by Rothman (47) is similar. Another possibility is that the sorting of membrane proteins is carried out by budding in the transitional region of the ER membrane in which transfer vesicles arise. That is, membrane proteins destined for the Golgi apparatus and plasma membrane are collected selectively and ER membrane proteins are excluded from the region (33). This possibility is supported by the absence of cytochrome P-450 on the cytoplasmic surface of the membranes of both the transfer vesicles and the Golgi cisternae. A similar sorting mechanism may work in the last trans-Golgi cisternae where secretory vesicles and lysosomes are formed. This sorting mechanism appears to be much more complicated because of the probable recycling of membrane proteins among the plasma membrane, lysosomes and Golgi apparatus. Intracellular transport of the membrane protein in the outer nuclear membrane, the ER, the Golgi apparatus, secretory vesicles and the plasma membrane is summa- rized as follows : Subcellular Compartments 103

Probable sorting sites of membrane proteins are indicated by asterisks and transport routes still in dispute by broken lines (see also Fig. 5). The properties of these M2 membranes appear to change markedly during transport through the Golgi apparatus, in which membrane proteins are glycosylated terminally. In chart shown we call the membranes of the organelles before the Golgi apparatus "preGolgi membranes" and those after "postGolgi membranes". Membranes in each subgroup can fuse with each other but not with those of the other subgroups. Thus, in formation of autolysosomes, the ER membrane would first be enclosed by an isolation membrane (probably derived from postGolgi membrane), the autophagosomes formed could then fuse with the lysosomes (32) to become autolysosomes.

IX. Phylogenesis of eukaryotic cells and subcellular compartments The subcellular compartments of prokaryotic and eukaryotic cells have been discussed, and it is interesting that the complicated compartmentation described here is quite common to all eukaryotic cells in various stages of phylogenetic develop- ment. This means that the formation of complicated subcellular compartments is important for the phylogenetic development of eukaryotic cells. Two hypotheses have been proposed to exaplain the origin of eukaryotic cells and their organelles, particulary mitochondria and chloroplasts : One is autogenesis and the other endosymbiosis. The former hypothesis (55) postulates genome duplication and invagination of the plasma membrane to form double membrane structures around each genome and that intracellular organelles are produced by a gradual and con- tinuous evolutionary process ; the nuclear genome becoming enlarged and differ- entiated, and the organellar genomes of the mitochondria and chloroplasts specialized by the lose of many duplicated cistrons. According to the second hypothesis, the mitochondria and chloroplasts originate from the invasion of a bacterium-like and a blue green alga-like organism into pre-eukaryote anaerobic type cells (28). These two hypotheses are competitive, and much of the data adduced so far in favor of one hypothesis is equally supportive of the other. Possible compartmentalization processes in the phylogenesis of eukaryotic cells according to the autogeneous (I) and endosymbiotic hypotheses (II) are shown in Fig. 7. It is notable that only organelles containing genomes have double membrane structures. Both hypotheses predict that all the odd-numbered spaces (S3, S5) in the present eukaryotic cells had been connected to the extracellular space sometime in the long history of phylogenic development. It is interesting to point out here that Robertson 104 Y. Tashiro

Fig. 7. Possible compartmentalization of a eukaryotic cell based on the autogeneous (I) and symbiotic (II) hypothesis of the phylogenesis of eukaryotic cells. In this figure (C.D.), the inter- membrane space of the mitochondria and chloroplasts purposely are shown as continuous to the extracellular space to clearly illustrate the origin of these organelles. Probably these connections are lost in the early stage of development (E). Subcellular Compartments 105

(44) made similar interpretation based on the assumption that most of the organelles are formed from the unit membrane structure bounding all the cells. He suggested that the luminal spaces of all the organelles are continuous with the extracellular space. This interpretation was accepted and even introduced in a number of text books. It is now believed that these spaces should not be continuous with the extra- cellular space. Introduction of a channel protein, such as mitochondrial porin, may have markedly changed the microenvironment of the intermembrane space so as to make the mutual communication among the semiautonomous organelles and the surrounding cyto- plasm much easier. The establishment of the discontinuity between S3 and S 1 between S3-ER and S3-GO or between S3-GO and S1 should have a strong effect on the evolution of cytoplasmic organelles in eukaryotic cells, by making possible the sorting of special marker enzymes during the intracellular transport of membrane proteins, thus facilitating differentiation of various cytoplasmic organelles.

Acknowledgments.I thankDr. T. Kanasekiof the TokyoMetropolitan Institute for Neuroscience and Drs. S. Matsuura,K. Omori,H. Nakada and R. Masakiof our departmentfor their pertinent commentson the text and MissK. Miki for her assistancewith the manuscript.

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(Received for publication, April 15, 1983)