Subcompartments of the endoplasmic reticulum

seminars in

CELL BIOLOGY, Vol 3, 1992 : pp 325-341

Subcompartments of the endoplasmic reticulum Barbara M . Vertel, Linda M. Walters, and David Mills The endoplasmic reticulum (ER) is the largest continuous endomembrane structure in the cytoplasm . It may be viewed as a series of unique subcompartments . In this review, we examine the rough ER, nuclear envelope and several smooth ER subcompartments. Consideration is given to the characteristic properties and functions of the ER and its domains, and to the formation and maintenance of subcompartments . Associations within the ER membrane bilayer, and with constituents of the cytoplasm and the ER lumen, contribute to the formation of domains and lead to the establishment of subcompartments that reflect specialized functions and vary according to the physiologic state and phenotype of the individual cell . Although the structural complexity of some ER subcompartments (such as the sarcoplasmic reticulum) is highly elaborate, the ER remains a dynamic organelle, subject to assembly and disassembly, capable of extensive remodelling and active in exchange with other organelles through mechanisms of membrane transport . Key words : endoplasmic reticulum / rough ER / smooth ER / nuclear envelope / intermediate compartment / calcium regulation / ER degradation

THE ENDOPLASMIC RETICULUM (ER) courses

Figure 1 . Diagram of ER subcompartments . The nuclear envelope (ne), rough ER (rER), smooth ER (sER), transitional ER (tER), and intermediate compartments (ic) are indicated . Asterisks identify the rough ER lumen . Also noted are nuclear pore complexes (npc), nucleus (nu), cis-Golgi (cG) and trans-Golgi (tG) .

through the cytoplasm of eukaryotic cells as a threedimensional network of continuous tubules and sheets . It is the first and largest in the series of cytoplasmic endomembrane compartments . A highly dynamic structure, the ER is involved in the synthesis and processing of secreted proteins, membrane proteins, and organelle resident proteins . It is a site of lipid synthesis, metabolism and detoxification reactions as well . Recent studies highlight the importance of the ER in the assembly, sorting, and degradation of proteins, the regulation of intracellular calcium and the presentation of antigen . Although these diverse functions are performed within a continuous, membrane-limited structure, subcompartments of the ER are also recognized (Figure 1) . 1-3 Utilization

of specific ER subcompartments, and variability in their morphological and biochemical characteristics, reflect the specialized function and phenotypic expression of individual cells . Currently we view the Golgi as a multicompartment organelle involved in the post-translational processing of proteins, and the sorting of biosynthetic products and recycling membrane traffic . 4,5 It is the purpose of this review to look at the ER from a similar perspective . In this context, we will examine the rough ER, the nuclear envelope and several subcompartments of the smooth ER . Discussion will be directed toward structure-function relationships in the formation and maintenance of subcompartments . The rough ER

From the Department of Cell Biology and Anatomy, The Chicago Medical School, 3333 Green Bay Road, North Chicago,

The presence of membrane-associated ribosomes distinguishes the rough ER easily from the smooth ER (Figure 2) . As a result of this association,

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Figure 2 . Electron micrograph of the rough and smooth ER of a rat hepatocyte . Magnification x 75600 . Graciously provided by George Palade . translation and cotranslational activities are largely restricted to the rough ER subcompartment . For those proteins directed to the ER, associations begin in the cytosol, where ribosomes, mRNA, and the nascent polypeptide form a complex with a ribonucleoprotein signal recognition particle . The complex then interacts with signal recognition particle receptor and probably other proteins (e .g . the signal sequence receptor) 6 located in the membrane of the rough ER to form higher-order complexes that function in the translocation of the nascent peptide . Other proteins implicated in ribosome attachment or translocation, such as the ribophorins, are similarly concentrated in the rough ER membrane . 7 ' 8 After formation of the translocation complex, translation continues and the nascent peptide is translocated across the rough ER membrane . 9 " 10 Within the lumen, the hydrophobic signal peptide is cleaved by signal peptidase, and N-asparagine-linked oligosaccharides are added cotranslationally from a dolichol phosphate intermediate embedded in the ER membrane . N-glycosylation involves a highly coordinated series of events requiring temporal and spatial organization, exposure of peptide glycosylation sites, and the availability of completed

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dolichol intermediates, sugar nucleotides, transporters, and carbohydrate processing enzymes . 11-13 Subsequent modifications of the carbohydrate chains begin in the ER with trimming of glucose and some mannose residues, followed by continued processing in the Golgi where Endo H resistance is conferred . Thus, a subdomain is created around the translocating nascent protein . In many cases, alterations in N-glycosylation have been implicated in the malfolding and faulty oligomerization of secretory glycoproteins . 14,15 As a result, misfolded and incorrectly assembled proteins may be aggregated, retained, and sometimes degraded in the ER . It is thought that the positioning, and polarity or large size of N-linked oligosaccharides may be required for orientation during the initial stages of folding . 16 Correct folding is, for many proteins, an important prerequisite to the acquisition of native structure, oligomerization and transport from the ER . 3,17 These events may be initiated cotranslationally, and largely occur in the rough ER subcompartment . In studies of influenza hemagglutinin and vesicular stomatitis virus G proteins, folding was observed to occur very rapidly after synthesis, and was required for normal subunit assembly and subsequent transport to the Golgi . 16,18-2 ° Initial folding appears to be necessary for subunit recognition prior to complex formation . Additional folding steps involving assembling complexes are also required to achieve quaternary structure for final stabilization and transport to the Golgi . In contrast, unassembled subunits (either misfolded or excessively produced) and partially assembled complexes have varying fates : some are stably retained in the ER, some may be retained and subsequently degraded in the ER, and others are transported to the Golgi and sorted to lysosomes . 21 A number of resident ER proteins facilitate these processes . Chaperonins (such as binding protein (BiP), colligin, glucose-regulated protein (grp) 94 and T cell receptor-associated protein) are believed to guide protein folding by a series of associationdissociation reactions as the protein matures . 22 They may prevent premature aggregate formation and increase the efficiency of folding, thereby promoting oligomerization . Concentrations of chaperonins increase during the accumulation of aberrant proteins within the ER . Frequently, chaperonin association with misfolded proteins aids in their retention in the ER, and adds selectivity to the transport mechanism . 22,23 Without chaperonin

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association, a degradation signal or proteolytic site may be exposed and target the protein for ER degradation . Disulfide bond formation, catalyzed by the abundant ER resident protein, protein disulfide isomerase (PDI), is another important part of folding that often begins cotranslationally . 22,24,25 After synthesis and processing within the ER, fully mature secretory proteins are transported to the Golgi ; however, resident ER proteins are selectively retained . 26 A C-terminal KDEL or related peptide sequence has been identified as a specific retention signal for luminal ER proteins such as BiP, PDI and grp 94 . The observation that ER luminal proteins leave the ER, but are returned, suggested the recycling of resident ER proteins through a post-ER compartment where a KDEL-receptor is present . Resident proteins are retrieved from that compartment and returned to the ER by vesicular trafficking . 22,27-30 It is believed that additional mechanisms also operate to retain both membrane-spanning and luminal resident ER proteins . The functional complexes that direct and participate in the processes described above contribute to the establishment of the rough ER subcompartment . Interactions of proteins and protein complexes within the phospholipid bilayer of the ER membrane are involved, as are interactions with constituents of the cytosol, such as the translation complexes formed among the signal recognition particle, ribosome and nascent polypeptide . Interactions with products residing within the ER lumen are also critical . The recent focus on folding, oligomerization and other aspects of protein assembly emphasizes the importance of intraluminal events .

Are there restricted domains of the rough ER? It is reasonable to imagine that, in turn, similar assemblies and interactions create subcompartments within the rough ER itself. Pryme 31 reviewed restricted domains of the rough ER from the perspective of biochemical properties of subfractionated rough microsome preparations and concluded that subdomain differences exist with respect to the numbers of associated ribosomes and the content of RNA, cholesterol and phospholipid . He further suggested that functional differences among rough ER subdomains might involve the translation of different messages or subclasses of messages, the restricted synthesis of specific lipids and phospholipids, and variations in relation

327 to the cell cycle, interactions with the cytoskeleton, and interactions with mitochondria . The biosynthesis of some collagens requires the association of alpha chains synthesized from two different genes to form triple helical molecules, and it has been proposed that restricted domains of the rough ER are utilized at the level of cotranslation to facilitate chain selection and folding . 32,33 The precedent for localized concentrations of mRNAs involved in the synthesis of specific proteins has been set by Lawrence and Singer34 for actin biosynthesis, and the possibility that a similar process is utilized for the synthesis of endomembranetranslocated products should be considered . In one case, targeting of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase synthesis appears to be directed to the outer membrane of the nuclear envelope . 35,36 Nonetheless, for many oligomeric proteins synthesized by the ER, the association of polysomes or their products is not required prior to assembly . The synthesis and assembly of viral glycoproteins have been well studied in this regard . 3,19 Lectin localization studies support the view of heterogeneity within the rough ER . Nonhomogeneous reactions within the rough ER of some cells have been observed for lectins such as Helix pomatia, which recognizes N-acetylgalactosamine, and Pisum sativum and Lens culinaris which recognize other carbohydrate structures . 37-39 It is not clear whether these localization patterns reflect the nonhomogeneous distribution of selected biosynthetic products, the function of subcompartments committed to specific oligosaccharide modifications, or a combination of the two possibilities . The restricted condensation of specific protein aggregates in intracisternal granules has been described in the rough ER of hyperstimulated endocrine cells, 40 and in exocrine pancreatic cells . 41,42 Recently, Valetti et a1 43 investigated the origin of Russell bodies and demonstrated that mutant immunoglobulin aggregates were concentrated and retained within structures bounded by ribosome-studded membranes . Resident ER proteins such as BiP and PDI appear to be excluded from intracisternal granules 42 and Russell bodies . 43 Ultimately, intracisternal granules are destined for autophagic vacuoles, 44 while Russell bodies are not . Apparently, selective sequestration can be a part of cellular processes involved in the removal of abnormal concentrations of protein aggregates in the rough ER .

32 8 Finally, it has been shown recently that the rough ER is involved in the formation of autophagic vacuoles . 45,46 It will be of interest to know what signals initiate this process and whether or not specialized subdomains of the rough ER are utilized .

The nuclear envelope (NE) The NE is a specialized and critically important subdomain of the ER that defines the boundary between the nucleus and cytoplasm . 47,48 The establishment of separate nuclear and cytoplasmic compartments by the NE is a fundamental feature of the eukaryotic cell . Within the nucleus, DNA is replicated and RNA is transcribed and processed . Independently, proteins are translated from mRNAs in the cytoplasm . This segregation permits the regulation of gene expression at a multiplicity of levels . The NE is a double membrane structure perforated by numerous nuclear pores that serve as the sites of nucleocytoplasmic exchange . The inner nuclear membrane functions in close association with the nuclear lamina to provide a scaffolding for the structural organization of the nucleus and the attachment of interphase chromosomes . The outer membrane of the NE is continuous with the rough ER and shares similar features with it . The lumen of the NE is continuous with the lumen of the ER as well . Continuity of the phospholipid bilayer of the inner and outer nuclear membranes is suggested by ultrastructural observations that reveal characteristic trilaminar structures in regions directly interfacing the nuclear pore complex . Despite this apparent membrane continuity, the inner and outer nuclear membranes remain as discrete domains . Nuclear pore complexes are elaborate structural entities that mediate the selective transport of RNAs and proteins between the nucleus and cytoplasm, an important aspect of the regulation of gene expression . They span the nuclear envelope at points where the inner and outer nuclear membranes meet . The pore complexes exhibit a striking eightfold symmetry when viewed perpendicular to the NE, and a characteristic architecture whose basic features include a nuclear and cytoplasmic ring, and a central plug surrounded by central spokes . 49 Transported molecules move through an expandable aqueous channel in the central plug/spoke region . The nuclear pore complex is an impressive supramolecular structure that represents the assembly of numerous proteins and is associated directly with proteins of the NE in regions of contact .

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On the inner face of the inner nuclear membrane, interactions with the nuclear lamina lead to the construction of a nuclear scaffold . The scaffold is a fibrous meshwork principally comprised of the three major lamins, A, B and C, which are related to intermediate filaments . It has been proposed that chromatin-associated lamins A and C interact with inner nuclear membrane-associated lamin B in mammalian cells . 48 Recent studies in Xenopus suggest that a vesicle-bound receptor and perhaps additional components mediate interactions between the NE and chromatin, and that regulation involves phosphorylation and dephosphorylation . 5o ,51 The characteristic properties of the inner nuclear membrane distinguish it clearly as a specialized domain, despite the observed continuity of the phospholipid bilayer of the inner and outer nuclear membranes . One of the most interesting features of the NE is its reversible disassembly during mitosis and cell division . The vesicles of the NE that form during this process remain distinct from the vesicles formed from the rest of the ER and the Golgi . These observations strongly support the view that the NE is continuous with the rest of the ER, but is at the same time a unique subcompartment . Although the regulatory signals that allow homotypic vesicle fusion and prevent heterotypic vesicle fusion are not completely characterized, progress has been made using several models developed to investigate this important problem . Extracts of amphibian eggs, cultured mammalian cells and Drosophila embryos have been combined with chromatin substrates prepared from demembranated sperm nuclei, chromosomes and naked DNA to reconstitute cell-free systems capable of nuclear assembly . 48,5 o Whether or not in vivo nuclear assembly is accomplished the same way remains to be determined . 52 It is of interest to note that the vesicles forming from the NE during the disassembly process in Xenopus are uniformly 75 nm-the same size as the vesicles thought to mediate ER to Golgi and intra-Golgi translocations . Recent reports implicate GTP and GTP-binding proteins in nuclear vesicle fusion, 53,54 and may suggest the involvement of membrane coating ; however, no coats or coat proteins have been identified . Although continuity with the rough ER is well established for the outer, ribosome-associated membrane and the lumen of the NE, the existence of distinct subdomains is also suggested for these structures . In some cases, the immunolocalization of biosynthetic product is observed throughout the rough

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Figure 3 . Smooth ER cisternae, sinusoidal ER, and crystalloid ER in the same UT-1 cell . UT-1-cw cells were incubated in the presence of compactin for 72 h . Smooth ER membrane apposed to the nuclear envelope is in continuity with the sinusoidal ER membrane (open arrows) . The latter membrane is in continuity with the tubules of the crystalloid ER (solid arrows) . N, nucleus . Bar, 0 .5 µm . Reproduced from ref 36, with kind permission of the authors and the Rockefeller University Press . ER lumen, but absent from the lumen of the NE37,55,56 while in other cases, immunostaining is equivalent in all luminal regions . 57 Interestingly, the overproduction of HMG CoA reductase leads to the assembly of an elaborate crystalloid ER of smooth membranes that appears to be inserted specifically into the NE in the mammalian UT-1 cell line 35,36 (Figure 3) and in yeast . 58 Thus, the NE remains continuous with the rest of the ER and is also characterized by several distinctive domains that play essential roles in the basic functions of the eukaryotic cell . It is clear that associations exist between the NE and nuclear pore complexes, but the molecular basis of these associations is not yet known . Although the NE seems to be continuous between the inner and outer nuclear membranes, the point of contact at the nuclear pore complex appears to establish these as separate domains . The nucleoplasmic face of the inner nuclear membrane interacts specifically with lamins and perhaps other proteins in a manner that in some experimental systems is suggestive of receptor-mediated processes and subject to regulation by phosphorylation-dephosphorylation reactions during

the cell cycle . On the cytosolic side, the outer nuclear membrane shares similarities with the rough ER, yet some observations suggest there may be differences here as well . Nucleocytoplasmic transport via the nuclear pore complex and nuclear assembly are two areas of active investigation, and major progress is being made in our understanding of the mechanisms and regulation of these processes . The significance of highly structured NE subcompartments will be clarified in future studies .

The smooth ER The distinction between the ribosome-studded rough ER and the smooth ER is readily apparent (Figure 2) . Although the smooth ER has no ultrastructural features that allow the clear discrimination of separate domains within it, it is likely that it is comprised of a number of separate subcompartments that serve a range of different functions . As a consequence, variations in the extent and arrangement of the smooth ER reflect phenotypic and physiological differences among individual cells .

330 Several functions of the smooth ER and their structural correlates are examined in the sections below . In some cases, the structural complexity of subcompartments is quite elaborate and unique .

Classical functions of the smooth ER

The smooth ER is abundant in cells active in lipid metabolism, steroidogenesis, glycogen storage and breakdown, and the detoxification of harmful substances . 2,59 For example, in testicular Leydig cells, the smooth ER comprises 60% of the total membrane area60 and functions in the synthesis of cholesterol and its conversion to testosterone . Within the liver, hepatocytes (Figure 2) detoxify harmful substances through oxidation, hydroxylation or methylation reactions catalyzed by enzymes located in the smooth ER (e .g . the cytochrome P450s) . Other enzymes of the smooth ER, such as glucuronyl transferase, remove barbiturates, antihistamines or other toxic substances by conjugation with charged water-soluble molecules (sulfate or glucuronic acid), whereupon detoxified products are excreted in the urine . The amount of smooth ER in hepatocytes will increase rapidly when an animal is exposed to certain drugs such as phenobarbital, and this expansion is correlated with drug tolerance . 61 After exposure and removal of the drug, the amount of smooth ER decreases by mechanisms not currently known . Expansion of the smooth ER in response to drug exposure has been utilized to obtain an immunological probe for the smooth ER enzyme, epoxide hydrolase . 62 The modulation of this smooth ER compartment offers an interesting biological system for further studies of membrane dynamics and compartmentalization .

Calcium regulation

Biological systems that regulate calcium exhibit a range of morphological and biochemical complexities . In striated muscle, calcium regulates the cycles of contraction and relaxation within the sarcomere . The demand for localized, rapid and reversible release of intracellular calcium is accommodated by an elaboration of the smooth ER that is so extensive and dramatic that the compartment seems to assume the identity of unique organellethe sarcoplasmic reticulum (SR) . The SR is comprised of longitudinal cisternae surrounding

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myofibrils and terminal cisternae specifically associated with the T tubules of the sarcolemma . The separate subcompartments of the SR are concerned with different aspects of calcium regulation . Terminal cisternae function in signalling calcium release and storing high concentrations of calcium, while longitudinal cisternae function in the removal of calcium from the cytosol via calcium ATPase . 63 Complex structural assemblies incorporating cytosolic, SR membrane and luminal components are characteristic of the terminal cisternae . For example, the calcium release channel/ryanodine receptor is an oligomer of a single, high molecular weight polypeptide residing within the phospholipid bilayer of the terminal cisterna that faces the T tubules of the sarcolemma and exhibits an unusual foot-like structure that protrudes beyond the SR membrane on its cytosolic face in regions of contact with the T tubules . 64,65 Calcium stores are maintained within the SR by the low affinity, high capacity, calcium binding protein, calsequestrin, also located in the terminal cisternae . 66-6 s In freeze-fracture deep-etch studies, Franzini-Armstrong et a1 69 described within the terminal cisternae a threedimensional network of calsequestrin anchored preferentially to the portion of the SR membrane associated with the T tubules . Furthermore, an indirect association between the luminal calsequestrin and the junctional membrane mediated by `joining strands' was suggested . In contrast, the calcium ATPase is abundant in the longitudinal cisternae of the SR, and excluded from the terminal cisternae . 67,70 The observation of a regular row of pits in the luminal leaflet where the longitudinal and terminal cisternae meet suggests that the subcompartments are segregated . Thus, the intricate subdomains characteristic of the SR of striated muscle are maintained by a series of specific interactions involving a number of cytosolic, luminal and integral membrane proteins organized into highly ordered structures . They are assembled in response to stringent physiologic requirements for the segregation of rapid calcium release and re-uptake, and regulated by signal transduction at the membrane interface of terminal cisternae of the SR and T tubules of the sarcolemma . In non-muscle cells, too, important cellular events are signalled by the mobilization of calcium from intracellular stores . The discovery that inositol (1,4,5) triphosphate (IP3)-induced calcium release activity was associated with microsomes strongly favored consideration of the ER, or a part of the ER, as the

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signal-responsive, calcium storage organelle . 71 Although all of the components required for calcium regulation-receptors to signal calcium release, low affinity, high capacity calcium binding proteins, and calcium ATPases-have been demonstrated in nonmuscle cells, their precise organization within cytoplasmic structures is unclear . 72,73 The story may be complicated by the fact that only a part of the available calcium is released by IP3 signalling, suggesting the existence of two independently mobilized calcium pools . Nonetheless, a more integrated view is beginning to emerge . The Purkinje cell of the cerebellum, which has a significant requirement for calcium regulatory mechanisms, an unusual abundance of IP3 receptors, and an interesting variety of smooth membrane-limited organelles, has become a preferred model for immunolocalization studies (Figure 4) . 7 2, 75 The immunolocalization of IP3 receptors in the unusual smooth ER membrane stacks and other smooth surfaced ER tubules of the Purkinje cell remains the only successful detection of IP3 receptors in nonmuscle cells . The location of other calcium regulatory components was not determined in the initial reports because available antibodies did not recognize calcium binding protein in the rat cerebellum . Recently this difficulty was overcome when it was discovered that calsequestrin is utilized as the high capacity, low affinity calcium binding protein in chicken Purkinje cells . 74,75 New studies demonstrate that calsequestrin and calcium ATPase are present in regular ER cisternae, but may be highly concentrated in smooth

membrane-bound structures, which, in previous studies of liver, pancreas and HL-60 cells, were named 'calciosomes' to connote calcium storage organelles . 76 Other recent studies provide evidence for the existence of ryanodine receptor/calcium release channels in Purkinje cells as well . 77 The interesting, and perhaps surprising, distribution of calcium regulatory components has led to the proposal that it is the dynamic interactions among these organelles and subcompartments that serve to modulate intracellular calcium . 72,73 The entire ER may also be implicated in calcium regulation . Recent studies suggest that the localized mobilization of calcium may be important for folding, oligomerization and assembly processes, association with chaperonins and other activities within the lumen of the rough ER, and for coat formation and budding events leading to vesicular transport . 78-80 In this regard, a number of the resident ER proteins (e .g . BiP, grp 94, PDI) are capable of low affinity calcium binding . 78,8 ' Recently, a protein similar to cyclophilin (potentially involved in calcium-mediated signal transduction processes) was reported to be colocalized with the calcium binding protein calreticulin, and to a lesser extent, with the microsomal calcium ATPase . 82 Future studies will clarify the structural correlates and mechanisms of calcium regulation in non-muscle cells . Thus for many cells, the `generic' ER, and possibly other structures and ER subcompartments, may be involved in calcium regulation in ways yet

Figure 4 . Stacks of smooth ER cisternae in cerebellar Purkinje cells . A . A smooth ER cisternal stack closely apposed to the plasmalemma of a Purkinje cell dendrite . B . Continuity between one stack and the rough ER . Regularly spaced dense projections appear to connect apposed cisternae in the stack, and may also be seen at the surface of isolated ER tubules . The projections are slightly smaller than ribosomes, and less electron dense . Immunocytochemical results have suggested that these projections represent IP 3 receptor complexes . 74 75 Bars, 0 .2 µ.m . Figure 4A was generously provided by Kohji Takei and Pietro De Camilli . Figure 4B is reprinted from ref 74 with kind permission of the authors and the journal of Neuroscience Press .

3 32 to be determined . In specialized cells with higher and more specific demands for calcium regulation, however, the morphological and biochemical complexities of ER subcompartments are a little better understood . For example, the smooth ER of striated muscle cells is elaborately expanded and subcompartmentalized to form the sarcoplasmic reticulum . In Purkinje and several other types of non-muscle cells, combinations of smooth membrane-bound/smooth ER structures have been described . In a general sense, the characteristics of smooth ER specializations committed to calcium regulation reflect specific cellular demands . Further clarification will require the combination of sensitive measurements of localized calcium concentrations, in situ colocalization of regulatory molecules, and biochemical characterization of specific subcompartments . 72,73 It is anticipated that the emerging story will teach us new lessons about the creation and utilization of ER subcompartments .

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the transitional elements (Figure 5) . Biochemical studies utilizing temperature blocks suggested that the compartment was involved in the O-linked addition of N-acetyl galactosamine to serine and threonine . 87 In studies of mutant low density lipoprotein receptors, Pathak et a1 88 reported O-linked sugar modifications on proteins that progressed no further in the secretory pathway than the ER transitional elements . Perhaps along similar lines, Kabcenell and Atkinson89 implicated a late ER compartment in oligosaccharide trimming . We have observed the accumulation of chondroitin sulfate proteoglycan precursors in smooth membranelimited, tubulo-vesicular structures continuous with

Late ER, transitional ER, the intermediate compartment and the cis-Golgi network (CGN) The transitional ER, described by Palade and colleagues as a series of smooth membrane-limited buds and vesicles arising in transitional regions of part rough/part smooth ER, was viewed as the site of departure for biosynthetic products en route to the Golgi .' This segment of the translocation pathway, between the ER and Golgi, has sparked considerable interest, primarily due to the discovery of the retention signal for resident ER proteins, the provocative results generated by studies using the fungal agent Brefeldin A (BFA), 83,84 and the postulated functions of a salvage, recycling, or intermediate compartment . It has become increasingly difficult to determine where the ER ends and where the Golgi begins . The concept of the CGN, 85 to encompass both anterograde and retrograde vesicular traffic between the ER and the cis-Golgi, has emerged in response to this dilemma (see also Saraste and Kuismanen, 86 this issue) . The expansion of smooth ER in some cells suggests the existence of post-rough ER compartments, perhaps related to the transitional ER, that can be exploited for specialized function . Several lines of evidence suggest that one such compartment mediates oligosaccharide modification . Tooze et a1 87 described a budding compartment for the mouse hepatitis virus-A59 lying between the rough ER and cis-Golgi and bearing morphological features characteristic of

Figure 5 . The budding compartment in sac(-) cells at 6-7 h after infection with MHV-A59 . The smoothmembraned budding compartment with progeny virions and budding figures (arrows) is closely associated with cisternae of the rough ER, including transitional elements (te), which at this early stage of infection are not a site of virus budding . This example was selected to show budding compartments not juxtaposed to Golgi stacks, and therefore is comparable to the budding compartment and its distension by the accumulating virions . Frequently parts of the cytoplasmic face of the budding compartment have a `bristle' coat and appear to be sites of fusion of small vesicles (arrowheads) . Bar, 0 .25 µm . Reprinted from ref 87 with kind permission of the authors and the Rockefeller University Press .

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ribosome-studded regions of the rough ER of cultured chicken chondrocytes 5 fi (Figure 6) . It is possible that this compartment functions in oligosaccharide modifications, such as the O-linked addition of xylose and perhaps additional linkage sugars required for the covalent attachment of glycosaminoglycan chains, or that it performs other functions concerned with proteoglycan synthesis, processing or assembly . In a related line of work, we have investigated the avian genetic mutant, nanomelia . This lethal mutation is characterized by severe skeletal abnormalities resulting from the defective production of cartilage proteoglycan . Studies demonstrated that nanomelic chondrocytes fail to complete the synthesis and processing of the cartilage chondroitin sulfate proteoglycan, and instead produce a truncated core protein precursor that, based on pulse-chase labeling studies, appears to be degraded intracellularly . 90 Interestingly, the mutant chondrocytes accumulate the truncated precursor in smooth membrane-limited regions continuous with the rough ER . 91 Other studies have suggested that phosphorylation of mannose-residues bound to lysosomal enzymes 22,92 and palmitoylation 93 occur in a late ER, preGolgi compartment . In some cases, the function of an expanded smooth ER compartment is less clear . For example, Rizzolo et a1 94 described the elaboration of smooth membrane extensions of modified ER cisternae in cells synthesizing chimeric growth hormone/viral envelope glycoproteins . Recently, the accumulation of rubella virus E l glycoprotein in a tubular, smooth ER of transfected COS cells was observed . 9 ' Some of the examples discussed above may represent expansions of a smooth membrane-limited compartment lying further along the secretory pathway, corresponding to an intermediate or salvage compartment . This compartment has been the subject of intensive investigation . One of the first indications of its existence came from experiments using low temperature blocks . Presumably, the kinetic block to vesicular transport leads to the backup of Golgi traffic and expansion of a vacuolar compartment proximal to the cis-Golgi . 96,97 It has been proposed that recycling of resident ER proteins via the KDELreceptor also occurs in this compartment ; hence its alternative identity as a salvage compartment . 22,28,29 In the presence of BFA, anterograde transport from the ER to the Golgi is prevented, while retrograde traffic remains active . Apparently, BFA causes membrane vesicles to uncoat (see below) . As a result, Golgi and intermediate membrane compartments tubulate and,

333 with time, collapse down onto the ER/intermediate compartments . The recent demonstration of BFA effects on membrane compartments of the trans-Golgi network, and even lysosomes emphasizes the importance of interrelationships among membrane compartments . 83,84 In light of these findings, the distinction between the ER/intermediate compartment(s)/Golgi (CGN) and the trans-Golgi network is validated, but within each of these membrane systems, the independence of discrete cisternal compartments becomes less clear . Nonetheless, the observation that an expanded smooth ER network (continuous with the regular ER) forms in retrovirustransformed murine erythroleukemia cells treated with BFA, and is used preferentially for virion budding would suggest that subcompartments of the ER can be maintained even in the presence of BFA . 98 The assessment of similarities and differences among these various compartments awaits further investigation . Our appreciation of the three-dimensional morphology of these fascinating structures and an evaluation of whether or not they are directly continuous with the ER, cis-Golgi and other tubulo-vesicular structures is hampered by the limitations inherent in the use of thin sectioned materials for ultrastructural analysis, though some serial section analyses are helpful in this regard 56,87,96 (Figure 7) . High voltage electron microscopic studies of thick sections have provided integrated views of structures like the Golgi, 99 and a similar approach might improve our understanding of structural relationships of compartments between the ER and Golgi . A detailed characterization of these compartments has been greatly limited by the lack of specific markers . Recently, membrane trafficking and fractionation studies have benefitted from the localization of 53 kDa and 58 kDa membrane proteins in some of these intermediate subcompartments, and the availability of antibodies against these proteins . loo-103 A preliminary report indicating that a 63 kDa protein may be present not only in the 53 kDa-containing compartment, but also in membranes closer to the rough ER, is of particular interest because it suggests heterogeneity within these domains . 104 Progress in the isolation and biochemical characterization of these compartments has been slow, but subcellular fractionation of transitional vesicles 105 and the use of free-flow electrophoresis in isolation protocolsto 6 are encouraging developments . Other approaches that utilize cell-free systems and permeabilized cells in reconstitution

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Figure 6 . CSPG precursor-containing regions of the ER are smooth membrane-bound and continuous with the rough ER . A convergent tubular structure containing CSPG precursors is shown in this glutaraldehyde-fixed chondrocyte . The tubules are enclosed by smooth membrane, but are continuous with dilated or cisternal regions of the ER that are studded with ribosomes (asterisk) . Bar, 0 .25 µm . Reproduced from ref 90, with kind permission of the Rockefeller University Press . studies offer exciting possibilities for elucidating structure/function relationships in these compartments (reviewed in refs 80,107) . Without doubt, the characteristics of these organellar compartments are profoundly affected by vesicular trafficking between cisterns . Non-clathrin-coated vesicles mediate traffic from the ER to the Golgi and between Golgi stacks, and it is likely that vesicle coating and uncoating constitute an important regulatory mechanism for membrane budding and fusion . 80,83,107-109 In turn, vesicle formation controls the transfer of luminal contents and membrane proteins from one discontinuous cistern of the endomembrane system to another . This view is reflected in the large number of sec mutants identified in yeast genetic studies (see Franzusoff, 110 this issue) and in other studies that implicate GTP-binding proteins in the regulation of membrane trafficking . 109

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When vesicle coats are disrupted as a result of BFAtreatment, organellar membranes tubulate rather than vesiculate, fuse less discriminately, and generally compromise the integrity of separate organelles like the ER and Golgi . 83,84 The formation or expansion of an additional ER, post-ER, intermediate or pre-Golgi compartment could occur through a decrease in the rate of exiting vesicular transport, perhaps accompanied by an increase in the rate of entry into that compartment . 83,84 Additional membrane might arise from the mobilization of continuous lateral membranes of the rough ER or transitional ER, or from entry via vesicular transport (e .g . from retrograde movement) . Perhaps further expansion of the compartment normally involves tubular extensions accomplished through interactions with motors and cytoskeletal elements (see below), in a manner similar to that observed after BFA-treatments . Thus, the presence and extent of an expanded late ER or intermediate compartment could reflect a balance between the rates of entering and exiting vesicular traffic . Alternatively, biosynthesis processes could result in an increase in luminal contents and lead to localized subcompartment expansion . Perhaps the expansion observed during virion assembly occurs in this way (Figure 5) . Research activities in these areas are intense, and it is likely that our understanding of cellular processes occurring between the rough ER and early Golgi will be greatly influenced by insights that will emerge from these efforts over the next few years .

Dynamics of the endoplasmic reticulum The vesicular trafficking process described above represents one aspect of the dynamic interactions and rearrangements of the ER . Another involves the entire cytoplasmic ER network (reviewed in ref 111) . The form and distribution of the continuous ER tubules change dramatically within the living cell . The direct observation of these fascinating rearrangements in living cells has been accomplished through the incorporation of fluorescent dyes into the ER . These studies have revealed intimate relationships between ER cisternae and microtubules . 111,112 Membrane tubules extend, retract, and reextend along microtubules, fusing with other membrane tubules to form networks which are finally remodelled into reticular ER patterns . If microtubules are disrupted by nocodazole treatment, ER

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membranes collapse and aggregate . When the drug is removed, membrane tubules from the aggregate extend along repolymerizing microtubules . The elaboration and movement of these membrane tubules is likely to be modulated by interactions with one or more of the many possible motor proteins and may also involve other structural proteins . Studies using reconstituted in vitro systems prepared from cell extracts are addressing the energy, microtubule and motor requirements for ER dynamics . 113,114 Another aspect of ER dynamics involves modulation in response to changing functional demands . For example, the greatly increased rate of protein secretion in stimulated B cells is accommodated by the biogenesis of an expanded ER and preferential increases in certain resident ER proteins . 115 The dynamic view of ER mobility and biogenesis emphasized in these studies will influence our concepts about the formation and maintenance of ER subcompartments .

Is there a subcompartment for degradation? The synthetic and processing functions of the ER are complemented by quality control mechanisms for the identification and removal of abnormal proteins or protein complexes from the secretory pathway . Evidence suggests that cells have multiple proteolytic mechanisms available for eliminating faulty proteins and partially assembled complexes . The lysosomal pathway has been well characterized . 116 In this case, selectivity largely resides in the sorting of substrates to the lysosome . Autophagy, a second degradative process, involves the sequestration of cytoplasmic constituents by the ER and their subsequent degradation in lysosomes . 45,46 It has become increasingly clear that mechanisms for non-lysosomal degradation also exist within the cell . A unique process involving the selective retention and degradation of proteins within the ER has been the subject of many recent investigations . 117,118 ER-associated degradation provides an important mechanism for the rapid and selective elimination of abnormal proteins prior to transport to the Golgi . Membrane spanning, secreted, and resident ER proteins are all candidates for disposal by this mechanism ." 8 Multimeric complexes such as the T cell receptor and the asialoglycoprotein receptor, HMG CoA reductase, immunoglobulins, 119 and abnormal proteins implicated in several diseases such as Tay Sachs 120 and cystic fibrosis 121 are a few examples of proteins subject to ER degradation .

335 Detailed studies have established several important characteristics that distinguish ER degradation from other mechanisms of intracellular protein disposal . These include insensitivity to lysosomal inhibitors such as ammonium chloride and leupeptin, inhibition at low temperature, a high degree of substrate specificity, and extreme rapidity . 122 The rapid nature of the degradation, combined with the observation of immunostaining for the degraded species only within the ER, suggested the ER itself as the site of proteolysis . 57,122 Recent studies using normal and mutant chain variants of the T cell receptor have clarified several features of ER degradation . 118 A specific determinant present in the transmembrane domain was identified as a targeting signal for ER degradation . 123,124 Chimeric proteins containing this degradative sequence were then used in conjunction with a permeabilized cell system and subcellular fractionation to confirm the ER as the site of degradation . 125 Degradation of the chimeric proteins was independent of exogenous ATP and cytosolic factors . Similarly, the rough ER was identified as the site of degradation for the H2 subunit of human asialoglycoprotein receptor . In this case, an intermediate was generated by the initial degradative event . Interestingly, ATP depletion in this model system resulted in accumulation of the intermediate degradation product within the rough ER and inhibition of further degradation . 126,127 Recently, an ionophore-sensitive, calcium dependent, biphasic pattern of degradation was reported for luminally truncated variants of ribophorin 1 . 128 The authors suggested that rapid proteolysis in the rough ER was followed by transport of residual proteins to a second compartment where degradation was completed at an even greater rate . In morphological studies, we observed the accumulation of a mutant proteoglycan precursor targeted for degradation within a smooth membrane-enclosed subcompartment of the ER . 91 Yet another scenario has been described in muscle cells for the disposal of acetylcholinesterase (AchE) . All newly synthesized AchE molecules are transported to the early Golgi (as indicated by the acquisition of wheat germ agglutinin, but not ricin, lectin-binding properties) ; subsequently a subset of these molecules are transported via coated vesicles to the SR where they are rapidly degraded by a non-lysosomal mechanism . 129 Collectively, these data imply that variations in the mechanism and possibly the site of ER-associated degradation exist .

336 Many unanswered questions remain concerning ER degradation . Although transmembrane signals targeting degradation have been reported for a few membrane spanning proteins, targeting signals for luminal proteins remain a mystery . Specific details about the site and mechanisms of ER degradation need to be obtained for other systems . Preliminary data would suggest that variations will be revealed by further analysis . The possibility that ER degradation involves lateral movement within the ER to another subcompartment is not ruled out by existing studies . In view of the progress made in our understanding of ER degradation over the last several years, the prospects for future success are excellent .

How are subcompartments established and maintained? Subcompartments in the ER are established according to the principles that guide the formation of domains in model 'fluid/mosaic' biological membranes . 130 Within the phospholipid bilayer of the ER membrane, integral membrane proteins assemble to form oligomers and higher order complexes . Further associations may occur on the cytosolic or luminal faces of the membrane leading to the formation of extended complexes with very distinctive domains . For example, translocation complexes form on the cytosolic face of the rough ER . On the luminal side of the rough ER, interactions continue as the nascent protein undergoes glycosylation, folding and oligomerization, perhaps assisted in the process by resident luminal proteins such as the chaperonins (thereby forming a 'processing/conformation' domain) . Subdomains of the NE are created by associations of the inner nuclear membrane with lamins and chromatin, while interactions with nuclear pore complexes lead to the establishment of other characteristic subdomains . Terminal cisternae in the SR of striated muscle are constructed from ryanodine receptor/calcium channels in the membrane lipid bilayer which are likely to be tethered by interactions on the cytosolic face and may be complexed on the luminal face by a series of interactions that also involve calsequestrin . The maintenance of luminal subdomains poses a more difficult problem . It is clear that resident proteins of the ER lumen are retained while secretory products or membrane proteins targeted to other locations progress through the secretory pathway .

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Recently, retention has been ascribed to a dynamic recycling process that involves a membrane protein receptor interacting with a retention signal at the C-terminus of resident luminal proteins . It is probably the case that retention also involves other interactions of resident ER proteins within the lumen . In fact, it has been suggested that the extent of interactions within the rough ER lumen approaches the level of organization of a luminal matrix . Dynamic changes are characteristic of some ER subcompartments . Frequent modulations of form are so extensive in regions of the late ER to early Golgi, that some have concluded it is more useful to term the region the cis-Golgi network than to consider it as a series of separate subcompartments along the secretory pathway . For Purkinje cells, calcium regulation may involve a dynamic equilibrium of continuity and discontinuity among ER subcompartments and associated structures . In rapidly dividing cells, the distinct properties of the nuclear envelope are maintained throughout the assembly/ disassembly processes of mitosis . Even less obviously fluid ER subcompartments undergo rearrangements and remodelling . Lateral movements of constituents within the plane of the membrane or within the lumen are likely to occur, and may be followed by new interactions with peripheral structures on the cytosolic (or perhaps the luminal) side . Associations on the cytosolic face with motors and cytoskeletal structures such as microtubules are important for membrane mobility, and vesicular transport requires the assembly and disassembly of membrane coats . Thus, the ER is organized into distinct subdomains, but at the same time, plasticity is retained . A balance exists between plasticity and the stable maintenance of discrete subcompartments, though the specific dynamics will vary considerably for different ER subcompartments . Some, like the smooth membrane-limited late ER, and perhaps calcium regulatory subcompartments, may be subject to constant remodelling, whereas others, like the SR of striated muscle and the NE of slowly dividing cells, appear to remain relatively fixed in time and space, and undergo more minor modulations . The normal functioning of a eukaryotic cell is dependent upon subcompartments of the ER, the dynamic interrelationships among them, and their interrelationships with other organelles . The creation and maintenance of several ER subcompartments are fairly well understood, but for others, clarification awaits further research .

337

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Summary/future perspectives The ER is both a continuous, membrane-enclosed organelle and a series of distinctive subcompartments that are characterized by a wide range of architectural features and serve a multiplicity of specific functions . The properties of ER subcompartments reflect specialized demands, and vary according to the physiological state and degree of differentiation of each individual cell . Compartments may be built up into very elaborate structural entities and subsequently remodelled in response to the needs of the individual cell . Normally, a balance is maintained between organelle plasticity, the imposition of constraints leading to the establishment of localized subdomains, and the extent to which localized subdomains expand . The dynamic equilibria may vary for different regions of the ER, and at different stages in the life of a cell . The drastic effects of Brefeldin A on organelle integrity underscore the importance of maintaining that equilibrium for normal cell function . The present challenge is to understand how this balance is regulated . A key to our progress will be the elucidation of

molecular mechanisms that underly vesicular transport . We need to determine what signals are required for sorting, and what constitutes the selectivity of targeting . Research has been fruitful in this area, and answers are sure to be forthcoming . The recent discoveries that the ER or its subcompartments function in degradation, calcium regulation, and antigen processing raise new questions about ER structure, function, and organization . As other new ER activities are revealed, they, too must be understood in the overall context of this multifaceted organelle . The application of experimental approaches currently in place and the high level of interest in the subjects discussed in this review are likely to lead us to new insights . The ability to isolate, immunopurify, and biochemically characterize membrane subcompartments and associated molecules is essential, and new methods becoming available appear to be quite useful . Genetic studies in yeast have greatly advanced our knowledge of organelle construction and membrane trafficking . In one case tested, yeast and mammalian cells behaved similarly with respect to the localized insertion of

Figure 7 . Serial sections through a budding compartment in an uninfected sac(-) cell . In uninfected cells this compartment (arrowheads) is found closely associated with transitional element regions of the rough ER (arrows) . Often the budding compartment occurs between transitional elements and Golgi stacks but sometimes, as this figure shows, it is not adjacent to a Golgi stack . In this situation, because of the absence of smooth cis-Golgi membranes, it is easier to trace the budding compartment through serial sections . Note the complex irregular morphology of the budding compartment and the absence of ribosomes from its membrane . The micrographs shown are of three sections (S4,5,7) from a continuous ribbon of 13 sections, each 50-60 nm thick . In this example, therefore, the budding compartment extended through a depth of --650 nm . All micrographs are at the same magnification . Bar, 0 .25 µm . Reprinted from ref 87, with kind permission of the authors and the Rockefeller University Press .

33 8 HMG CoA reductase into the nuclear envelope, perhaps indicating the potential of yeast systems for studies of ER subcompartments . Genetic studies with mammalian cells are progressing, and are of considerable interest as well . The use of cell-free systems such as reconstituted nuclear assembly systems and functional Golgi transport and processing systems has elucidated many features of organelle structure and function, and should continue to do so . Reconstituted microtubule/motor/membrane systems have revealed new information about the dynamics of membrane movement and remodelling, and a recent report suggests that this type of in vitro system may also be useful for studying the kinetics of movement within the lumen . 131 Semi-permeable cells offer another excellent model for characterizing the biological properties of organelle structure and function . Credibility ultimately resides in vivo, and the application of genetic engineering, transfection experiments, and the microinjection of proteins and antibodies provides a reliable check of proposed mechanisms in the living cell . As we have seen with monensin, and currently with Brefeldin A, perturbation of the biological system by the action of an inhibitor adds challenges that test our operating hypotheses . The next several years are likely to lead to a deeper understanding of the organization and function of the ER, and provide us with a new set of questions .

Acknowledgements We thank numerous colleagues for helpful discussions . We gratefully acknowledge John Keller, Edward Kuczmarski and Jaakko Saraste for critical comments on the manuscript, and especially thank Bonnie Grier for her assistance . We express our gratitude to George Palade, Kohji Takei, Pietro DeCamilli, Ravi Pathak, Richard Anderson and Sharon Tooze for generously contributing the micrographs shown in Figures 2-6 . Our apologies to all researchers whose original contributions were referenced only through reviews . The authors' research is supported by NIH grant DK2843 .

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