CONTROL COMPARTMENT OF THE CELL
After entering the ER, newly synthesized proteins attempt to fold with the assistance of chaperones and folding enzymes, and their folding status is monitored by chaperones and also enzymes (Table 49–6).
TABLE 49–6Some Chaperones and Enzymes Involved in Folding That Are Located in the Rough Endoplasmic Reticulum ||Download (.pdf) TABLE 49–6 Some Chaperones and Enzymes Involved in Folding That Are Located in the Rough Endoplasmic Reticulum
BiP (immunoglobulin heavy chain binding protein)
GRP94 (glucose-regulated protein)
PDI (protein disulfide isomerase)
PPI (peptidyl prolyl cis-trans isomerase)
The chaperone calnexin is a calcium-binding protein located in the ER membrane. This protein binds a wide variety of proteins, including major histocompatibility complex (MHC) antigens and a variety of plasma proteins. As described in Chapter 46, calnexin binds the monoglucosylated species of glycoproteins that occur during processing of glycoproteins, retaining them in the ER until the glycoprotein has folded properly. Calreticulin, which is also a calcium-binding protein, has properties similar to those of calnexin, but it is not membrane-bound. In addition to chaperones, two enzymes in the ER lumen are concerned with proper folding of proteins. Protein disulfide isomerase (PDI) promotes rapid formation and reshuffling of disulfide bonds until the correct set is achieved. Peptidyl prolyl isomerase (PPI) accelerates folding of proline-containing proteins by catalyzing the cis–trans isomerization of X-Pro bonds, where X is any amino acid residue.
Misfolded or incompletely folded proteins interact with chaperones, which retain them in the ER and prevent them from being exported to their final destinations. If such interactions continue for a prolonged period of time, the misfolded proteins are usually disposed of by endoplasmic reticulum-associated degradation (ERAD). This avoids a harmful build-up of misfolded proteins. In a number of genetic diseases, such as cystic fibrosis, retention of misfolded proteins occurs in the ER, and in some cases, the retained proteins still exhibit some functional activity. As discussed later in this Chapter, there is much current interest in finding drugs that will interact with such proteins and promote their correct folding and export out of the ER.
MISFOLDED PROTEINS UNDERGO ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION
Maintenance of homeostasis in the ER is important for normal cell function. Perturbation of the unique environment within the lumen of the ER (eg, by changes in ER Ca2+, alterations of redox status, exposure to various toxins or some viruses), can lead to reduced protein folding capacity and the accumulation of misfolded proteins. The accumulation of misfolded proteins in the ER is referred to as ER stress. The cell has evolved a mechanism termed the unfolded protein response (UPR) to sense the levels of misfolded proteins and initiate intracellular signaling mechanisms to compensate for the stress conditions and restore ER homeostasis. The UPR is initiated by ER stress sensors, which are transmembrane proteins embedded in the ER membrane. Activation of these stress sensors causes three principal effects: (1) transient inhibition of translation to reduce the amount of newly synthesized proteins, (2) induction of a transcription leading to increased expression of ER chaperones and to (3) increased synthesis of proteins involved in degradation of misfolded ER proteins (discussed below). Therefore, the UPR increases the ER folding capacity and prevents a buildup of unproductive and potentially toxic protein products, in addition to other responses to restore cellular homeostasis. However, if impairment of folding persists, cell death pathways (apoptosis) are activated. A more complete understanding of the UPR is likely to provide new approaches to treating diseases in which ER stress and defective protein folding occur (see Table 49–7).
TABLE 49–7Some Conformational Diseases That Are Caused by Abnormalities in Intracellular Transport of Specific Proteins and Enzymes due to Mutationsa ||Download (.pdf) TABLE 49–7 Some Conformational Diseases That Are Caused by Abnormalities in Intracellular Transport of Specific Proteins and Enzymes due to Mutationsa
|Disease ||Affected Protein |
|α1-Antitrypsin deficiency with liver disease ||α1-Antitrypsin |
|Chediak-Higashi syndrome ||Lysosomal trafficking regulator |
|Combined deficiency of factors V and VIII ||ERGIC53, a mannose-binding lectin |
|Cystic fibrosis ||CFTR |
|Diabetes mellitus [some cases] ||Insulin receptor (α-subunit) |
|Familial hypercholesterolemia, autosomal dominant ||LDL receptor |
|Gaucher disease ||β-Glucosidase |
|Hemophilia A and B ||Factors VIII and IX |
|Hereditary hemochromatosis ||HFE |
|Hermansky-Pudlak syndrome ||AP-3 adaptor complex β3A subunit |
|I-cell disease ||N-acetylglucosamine 1-phosphotransferase |
|Lowe oculocerebrorenal syndrome ||PIP2 5-phosphatase |
|Tay-Sachs disease ||β-Hexosaminidase |
|von Willebrand disease ||von Willebrand factor |
TABLE 49–8Some Types of Vesicles and Their Functions ||Download (.pdf) TABLE 49–8 Some Types of Vesicles and Their Functions
|Vesicle ||Function |
|COPI ||Involved in intra-GA transport and retrograde transport from the GA to the ER |
|COPII ||Involved in export from the ER to either ERGIC or the GA |
|Clathrin ||Involved in transport in post-GA locations including the PM, TGN and endosomes |
|Secretory vesicles ||Involved in regulated secretion from organs such as the pancreas (eg, secretion of insulin) |
|Vesicles from the TGN to the PM ||They carry proteins to the PM and are also involved in constitutive secretion |
Proteins that misfold in the ER degraded by the ERAD pathway (Figure 49–10). This occurs by selective transport of both luminal and membrane proteins back across the ER (retrotranslocation or dislocation) to enter proteasomes present in the cytosol. The energy for translocation appears to be at least partly supplied by p97, an AAA-ATPase (one of a family of ATPases Associated with various cellular Activities), the precise route by which the misfolded proteins pass back across the ER membrane has not yet been established. A number of candidates have been suggested as possible transmembrane channels for ERAD. These include Sec61, the complex responsible for protein entry into the ER, degradation in ER protein 1 (derlin1), and the ERAD E3 ligases, Hrd1 and Doa10. However, although it seems reasonable to assume that proteins must exit the ER via a membrane pore, there is, as yet, no definitive evidence that such a channel exists, and it is possible that a completely different mechanism is used. For example, it has been suggested that membrane perturbation processes similar to those leading to the formation of cytosolic lipid droplets, or caused by the action of rhomboid proteins, which regulate intermembrane proteolysis, may be involved.
Simplified scheme of the events in ERAD. A target protein which is misfolded undergoes retrograde transport through the ER membrane into the cytosol, where it is subjected to polyubiquitination. Following polyubiquitination, it enters a proteasome, inside which it is degraded to small peptides that exit and may have several fates. Liberated ubiquitin molecules are recycled. Several proteins, including Sec61, Derlin 1 and the ERAD E3 ligases, Hrd1 and Doa10, are potential ERAD channel candidates. However, there is no clear evidence to demonstrate that a channel exists and alternative mechanisms involving membrane perturbation have also been proposed.
Chaperones present in the lumen of the ER (eg, BiP) and in the cytosol help target misfolded proteins to proteasomes. Prior to entering proteasomes, most proteins are ubiquitinated (see the next paragraph) and are escorted to proteasomes by polyubiquitin-binding proteins. Ubiquitin ligases are present in the ER membrane.
Ubiquitin Is a Key Molecule in Protein Degradation
There are two major pathways of protein degradation in eukaryotes. One involves lysosomal proteases and does not require ATP, but the major pathway involves ubiquitin and is ATP-dependent. The ubiquitin pathway is particularly associated with disposal of misfolded proteins and regulatory enzymes that have short half-lives. Ubiquitin is known to be involved in diverse important physiologic processes including cell-cycle regulation (degradation of cyclins), DNA repair, inflammation and the immune response (see Chapter 52), muscle wasting, viral infections, and many others. Ubiquitin is a small (76 amino acids), highly conserved protein that plays a key role in marking various proteins for subsequent degradation in proteasomes. The mechanism of attachment of ubiquitin to a target protein (eg, a misfolded form of cystic fibrosis transmembrane conductance regulator [CFTR], the protein involved in the causation of cystic fibrosis; see Chapter 40) is shown in Figure 49–12 and involves three enzymes: an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are a number of types of conjugating enzymes, and, surprisingly, some hundreds of different ligases. It is the latter enzyme that confers substrate specificity. Once the molecule of ubiquitin is attached to the protein, a number of others are also attached, resulting in a polyubiquitinated target protein. It has been estimated that a minimum of four ubiquitin molecules must be attached to commit a target molecule to degradation in a proteasome. Ubiquitin can be cleaved from a target protein by deubiquitinating enzymes and the liberated ubiquitin can be reused.
Protein degradation in the proteasome. 1. The regulatory particle recognizes the ubiquitinated protein which are unfolded by ATPases present in the regulatory particles or caps. 2. Protease active sites in the core of the proteosome attack peptide bonds and degrade the protein. 3. Peptides are released into the cytosol for further degradation by cytosolic peptidases.
Sequence of reactions in addition of ubiquitin to a target protein. The C-terminal COO− group of ubiquitin (Ub) is first linked in a thioester bond to an SH group of the activating enzyme (E1). The activated ubiquitin is transferred to an SH group of the conjugating enzyme. The transfer of ubiquitin from E2 to an ε-amino group on a lysine of the target protein is then catalysed by a ligase enzyme. Additional rounds of ubiquitination then build up the polyubiquitin chain. (LYS Pr, target protein.)
Ubiquitinated Proteins Are Degraded in Proteasomes
Polyubiquitinated target proteins enter proteasomes located in the cytosol. Proteasomes are protein complexes with a relatively large cylindrical structure and are composed of four rings with a hollow core containing the protease active sites, and one or two caps or regulatory particles that recognize the polyubiquinated substrates and initiate degradation (Figure 49–11). Target proteins are unfolded by ATPases present in the proteasome caps. Proteasomes can hydrolyze a very wide variety of peptide bonds. Target proteins pass into the core to be degraded to small peptides, which then exit the proteasome to be further degraded by cytosolic peptidases. Both normally and abnormally folded proteins are substrates for the proteasome. Liberated ubiquitin molecules are recycled. The proteasome plays an important role in presenting small peptides produced by degradation of various viruses and other molecules to MHC class I molecules, a key step in antigen presentation to T lymphocytes.
TRANSPORT VESICLES ARE KEY PLAYERS IN INTRACELLULAR PROTEIN TRAFFIC
Proteins that are synthesized on membrane-bound polyribosomes and are destined for the GA or PM reach these sites inside transport vesicles. As indicated in Table 49–8, there are a number of different types of vesicles. Other types of vesicles may remain to be discovered.
Each vesicle has its own set of coat proteins. Clathrin is used in vesicles destined for exocytosis (see discussions of the LDL receptor in Chapters 25 and 26), in some of those carrying cargo to lysosomes. This protein consists of three interlocking spirals, which interact to form a lattice around the vesicle. COPI and COPII, the vesicles involved in retrograde transport (from the GA to the ER) and anterograde transport (from the ER to the GA), respectively, however, are clathrin-free. Transport and secretory vesicles carrying cargo from the GA to the PM are also clathrin-free. Here we focus mainly on COPII, COPI, and clathrin-coated vesicles. Each type has a different complement of proteins in its coat. For the sake of clarity, the non-clathrin-coated vesicles are referred to in this text as transport vesicles. The principles concerning assembly of these different types are generally similar, although some details of assembly for COPI and clathrin-coated vesicles differ from those for COPII (see below).
Model of Transport Vesicles Involves SNAREs & Other Factors
Vesicles lie at the heart of intracellular transport of many proteins. The use by Schekman and colleagues of genetic approaches for studying vesicles in yeast and the development by Rothman and colleagues of cell-free systems to study vesicle formation have been crucial in the understanding of the events involved in vesicle formation and transport. For instance, it is possible to observe, by electron microscopy, budding of vesicles from Golgi preparations incubated with cytosol, ATP and GTP-γ. The overall mechanism is complex, and involves a variety of cytosolic and membrane proteins, GTP, ATP, and accessory factors. Budding, tethering, docking, and membrane fusion are key steps in the life cycles of vesicles, with the GTP-binding proteins, Sar1, ARF, and Rab acting as molecular switches. Sar1 is the protein involved in step 1 of formation of COPII vesicles, whereas ARF is involved in the formation of COPI and clathrin-coated vesicles. The functions of the various proteins involved in vesicle processing and the abbreviations used are shown in Table 49–9.
There are common general steps in transport vesicle formation, vesicle targeting and fusion with a target membrane, irrespective of the membrane the vesicle forms from or its intracellular destination. The nature of the coat proteins, GTPases and targeting factors differ depending on where the vesicle forms from and its eventual destination. Anterograde transport from the ER to the Golgi involving COPII vesicles is the best studied example. The process can be considered to occur in eight steps (Figure 49–13). The basic concept is that each transport vesicle is loaded with specific cargo and also one or more v-SNARE proteins that direct targeting. Each target membrane bears one or more complementary t-SNARE proteins with which the former interact, mediating SNARE protein-dependent vesicle–membrane fusion. In addition, Rab proteins also help direct the vesicles to specific membranes and their tethering at a target membrane.
TABLE 49–9Some Factors Involved in the Formation of Non-Clathrin-Coated Vesicles and Their Transport ||Download (.pdf) TABLE 49–9 Some Factors Involved in the Formation of Non-Clathrin-Coated Vesicles and Their Transport
ARF: ADP-ribosylation factor, a GTPase involved in formation of COPI and also clathrin-coated vesicles.
Coat proteins: A family of proteins found in coated vesicles. Different transport vesicles have different complements of coat proteins.
NSF: N-ethylmaleimide-sensitive factor, an ATPase.
Sar1: A GTPase that plays a key role in assembly of COPII vesicles.
Sec12p: A guanine nucleotide exchange factor (GEF) that interconverts Sar1·GDP and Sar1·GTP.
α-SNAP: Soluble NSF attachment protein. Along with NSF, this protein is involved in dissociation of SNARE complexes.
SNARE: SNAP receptor. SNAREs are key molecules in the fusion of vesicles with acceptor membranes.
t-SNARE: Target SNARE.
v-SNARE: Vesicle SNARE.
Rab proteins: A family of Ras-related proteins (monomeric GTPases) first observed in rat brain. They are active when GTP is bound. Different Rab molecules dock different vesicles to acceptor membranes.
Rab effector proteins: A family of proteins that interact with Rab molecules; some act to tether vesicles to acceptor membranes.
Model of the steps in a round of anterograde transport involving COPII vesicles. Step 1: Sar1 is activated when GDP exchanged for GTP and it becomes embedded in the ER membrane to form a focal point for bud formation. Step 2: Coat proteins bind to Sar1·GTP and cargo proteins become enclosed inside the vesicles. Step 3: The bud pinches off, formatting a complete coated vesicle. Vesicles move through cells along microtubules or actin filaments. Step 4: The vesicle is uncoated when bound GTP is hydrolyzed to GDP by Sar1. Step 5: Rab molecules are attached to vesicles after switching of Rab.GDP to Rab.GTP, a specific GEF (see Table 49–9). Rab effector proteins on target membranes bind to Rab·GTP, tethering the vesicles to the target membrane. Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to form a four helix bundle which docks the vesicles and initiates fusion. Step 7: When the v- and t-SNARES are closely aligned, the vesicle fuses with the membrane and the contents are released. GTP is then hydrolyzed to GDP, and the Rab·GDP molecules are released into the cytosol. An ATPase (NSF) and α-SNAP (see Table 49–9) dissociate the four-helix bundle between the v- and t-SNARES so that they can be reused. Step 8: Rab and SNARE proteins are recycled for further rounds of vesicle fusion. (Adapted, with permission, from Rothman JE: Mechanisms of intracellular protein transport. Nature 1994;372:55.)
Step 1: Budding is initiated when Sar1 is activated when GTP is bound in exchange for GDP via the action of Sec12p (Table 49–9), switching it from a soluble to a membrane bound form by causing a conformational change which exposes a hydrophobic tail. Thus it becomes embedded in the ER membrane to form a focal point for vesicle assembly.
Step 2: Various coat proteins bind to Sar1·GTP. In turn, membrane cargo proteins bind to the coat proteins either directly or via intermediary proteins that attach to coat proteins, and they then become enclosed in their appropriate vesicles. Soluble cargo proteins bind to receptor regions inside the vesicles. A number of signal sequences on cargo molecules have been identified (Table 49–1). For example KDEL sequences direct certain ER-resident proteins in retrograde flow to the ER in COPI vesicles. Di-acidic sequences (eg, Asp-X-Glu, X = any amino acid) and short hydrophobic sequences on membrane proteins are involved in interactions with coat proteins of COPII vesicles. Not all cargo molecules have a sorting signal. Some highly abundant secretory proteins travel to various cellular destinations in transport vesicles by bulk flow; that is, they enter into transport vesicles at the same concentration that they occur in the organelle. However, it appears that most proteins are actively sorted (concentrated) into transport vesicles and bulk flow is used by only a select group of cargo proteins. Additional coat proteins are assembled to complete bud formation. Coat proteins promote budding, contribute to the curvature of buds and also help sort proteins.
Step 3: The bud pinches off, completing formation of the coated vesicle. The curvature of the ER membrane and protein-protein and protein-lipid interactions in the bud facilitate pinching off from ER exit sites. Vesicles move through cells along microtubules or along actin filaments.
Step 4: Coat disassembly (involving dissociation of Sar1 and the shell of coat proteins) follows hydrolysis of bound GTP to GDP by Sar1, promoted by a specific coat protein. Sar1 thus plays key roles in both assembly and dissociation of the coat proteins. GTP-γ-S (a nonhydrolyzable analog of GTP often used in investigations of the role of GTP in biochemical processes) blocks disassembly of the coat from coated vesicles, leading to a build-up of coated vesicles, facilitating their study. Uncoating is necessary for fusion to occur.
Step 5: Vesicle targeting is achieved by attachment of Rab molecules to vesicles. Rabs are a family of Ras-like proteins required in several steps of intracellular protein transport and also in regulated secretion and endocytosis. They are small monomeric GTPases that attach to the cytosolic faces of budding vesicles in the GTP-bound state and are also present on acceptor membranes. Rab·GDP molecules in the cytosol are switched to Rab·GTP molecules by a specific GEF (Table 49–9). Rab effector proteins on target membranes bind to Rab·GTP, but not Rab.GDP molecules, thus tethering the vesicles to the membranes.
Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to dock the vesicles and initiate fusion. Generally, one v-SNARE in the vesicle pairs with three t-SNAREs on the acceptor membrane to form a tight four-helix bundle. In synaptic vesicles one v-SNARE is designated synaptobrevin. Botulinum B toxin is one of the most lethal toxins known and the most serious cause of food poisoning. One component of this toxin is a protease that binds synaptobrevin, thus inhibiting release of acetylcholine at the neuromuscular junction and possibly proving fatal.
Step 7: Fusion of the vesicle with the acceptor membrane occurs once the v- and t-SNARES are closely aligned. After vesicle fusion and release of contents occurs, GTP is hydrolyzed to GDP, and the Rab·GDP molecules are released into the cytosol. When a SNARE on one membrane interacts with a SNARE on another membrane, linking the two, this is referred to as a trans-SNARE complex or a SNARE pin. Interactions of SNARES on the same membrane form a cis-SNARE complex. In order to dissociate the four-helix bundle between the v- and t-SNARES so that they can be reused, two additional proteins are required. These are an ATPase (NSF) and α-SNAP (see Table 49–9). NSF hydrolyzes ATP and the energy released dissociates the four-helix bundle making the SNARE proteins available for another round of membrane fusion.
Step 8: Certain components, such as the Rab and SNARE proteins, are recycled for subsequent rounds of vesicle fusion.
During the above cycle, SNARES, tethering proteins, Rab, and other proteins all collaborate to deliver a vesicle and its contents to the appropriate site.
Some Transport Vesicles Travel via the Trans Golgi Network
Proteins in the apical or basolateral areas of the plasma membranes of polarized epithelial cells can be transported to these sites in transport vesicles budding from the trans Golgi network. Different Rab proteins likely direct some vesicles to apical regions and others to basolateral regions. In certain cells, proteins are first directed to the basolateral membrane, then endocytosed and transported across the cell by transcytosis to the apical region. Yet another mechanism for sorting proteins to the apical region (or in some cases to the basolateral region) involves the glycosylphosphatidylinositol (GPI) anchor described in Chapter 46. This structure is also often present in lipid rafts (see Chapter 40).
Once proteins in the secretory pathway reach the cis-Golgi from the ER in vesicles, they can travel through the GA to the trans-Golgi in vesicles, or by a process called cisternal maturation, in which the cisternae move and transform into one another, or perhaps in some cases diffusion via intracisternal connections that have been observed in some cell types. In this model, vesicular elements from the ER fuse with one another to help form the cis-Golgi, which in turn can move forward to become the medial Golgi, etc. COPI vesicles return Golgi enzymes (eg, glycosyltransferases) back from distal cisternae of the GA to more proximal (eg, cis) cisternae.
The Formation of COPI Vesicles Is Inhibited by Brefeldin
The fungal metabolite brefeldin A prevents GTP from binding to ARF, and thus inhibits formation of COPI vesicles. In its presence, the Golgi apparatus appears to collapse into the ER. It may do this by inhibiting the GEF involved in formation of COPI vesicles. Brefeldin A has thus proven to be a useful tool for examining some aspects of Golgi structure and function.
Some Proteins Undergo Further Processing While Inside Vesicles
Some proteins are subjected to further processing by proteolysis while inside either transport or secretory vesicles. For example, albumin is synthesized by hepatocytes as preproalbumin (see Chapter 52). Its signal peptide is removed, converting it to proalbumin. In turn, proalbumin, while inside secretory vesicles, is converted to albumin by action of furin (Figure 49–14). This enzyme cleaves a hexapeptide from proalbumin immediately C-terminal to a dibasic amino acid site (ArgArg). The resulting mature albumin is secreted into the plasma. Hormones such as insulin (see Chapter 41) are subjected to similar proteolytic cleavages while inside secretory vesicles.
Processing of preproalbumin albumin. The signal peptide is removed from preproalbumin as it moves into the ER. Furin cleaves proalbumin at the C-terminal end of a basic dipeptide (ArgArg) while the protein is inside the secretory vesicle. The mature albumin is secreted into the plasma.
THE ASSEMBLY OF MEMBRANES IS COMPLEX
There are a number of different types of cell membranes, ranging from the plasma membrane which separates the cell contents from the external environment to the internal membranes of subcellular organelles such a mitochondria and the ER. Although the general lipid bilayer structure is similar in all membranes, they differ in their specific protein and lipid content and each type has its own specific features (see Chapter 40). No satisfactory scheme describing the assembly of any one of these membranes is currently available. Vesicular transport and the way in which various proteins are initially inserted into the membrane of the ER have been discussed above. Some general points about membrane assembly are addressed below.
Asymmetry of Both Proteins & Lipids Is Maintained During Membrane Assembly
Vesicles formed from membranes of the ER and Golgi apparatus, either naturally or pinched off by homogenization, exhibit transverse asymmetries of both lipid and protein. These asymmetries are maintained during fusion of transport vesicles with the plasma membrane. The inside of the vesicles after fusion becomes the outside of the plasma membrane, and the cytoplasmic side of the vesicles remains the cytoplasmic side of the membrane (Figure 49–15). Phospholipids are the major class of lipid in membranes. The enzymes responsible for the synthesis of phospholipids reside in the cytoplasmic surface of the cisternae (the sac-like structures) of the ER. As phospholipids are synthesized at that site, they probably self-assemble into thermodynamically stable bimolecular layers, thereby expanding the membrane and perhaps promoting the detachment of so-called lipid vesicles from it. It has been proposed that these vesicles travel to other sites, donating their lipids to other membranes. Cytosolic proteins that take up phospholipids from one membrane and release them to another (ie, phospholipid exchange proteins) have been demonstrated; they probably play a role in contributing to the specific lipid composition of various membranes.
It should be noted that the lipid compositions of the ER, Golgi, and plasma membrane differ, the latter two membranes containing higher amounts of cholesterol, sphingomyelin, and glycosphingolipids, and less phosphoglycerides than does the ER. Sphingolipids pack more densely in membranes than do phosphoglycerides. These differences affect the structures and functions of membranes. For example, the thickness of the bilayer of the GA and PM is greater than that of the ER, which affects which particular transmembrane proteins are found in these organelles. Also, lipid rafts (see Chapter 40) are believed to be formed in the GA.
Fusion of a vesicle with the plasma membrane preserves the orientation of any integral proteins embedded in the vesicle bilayer. Initially, the amino terminal of the protein faces the lumen, or inner cavity, of such a vesicle. After fusion, the amino terminal is on the exterior surface of the plasma membrane. The lumen of a vesicle and the outside of the cell are topologically equivalent. (Redrawn and modified, with permission, from Lodish HF, Rothman JE: The assembly of cell membranes. Sci Am [Jan] 1979;240:43.)
Lipids & Proteins Undergo Turnover at Different Rates in Different Membranes
It has been shown that the half-lives of the lipids of the ER membranes of rat liver are generally shorter than those of its proteins, so that the turnover rates of lipids and proteins are independent. Indeed, different lipids have been found to have different half-lives. Furthermore, the half-lives of the proteins of these membranes vary widely, some exhibiting short (hours) and others long (days) half-lives. Thus, individual lipids and proteins of the ER membranes appear to be inserted into it relatively independently and this is believed to be the case for many other membranes.
The biogenesis of membranes is thus a complex process about which much remains to be learned. One indication of the complexity involved is to consider the number of posttranslational modifications that membrane proteins may be subjected to prior to attaining their mature state. These include disulfide formation, proteolysis, assembly into mul-timers, glycosylation, addition of a glycophosphatidylinositol (GPI) anchor, sulfation on tyrosine or carbohydrate moieties, phosphorylation, acylation, and prenylation—a list that is not complete. Nevertheless, significant progress has been made; Table 49–10 summarizes some of the major features of membrane assembly that have emerged to date.
TABLE 49–10Some Major Features of Membrane Assembly ||Download (.pdf) TABLE 49–10 Some Major Features of Membrane Assembly
Lipids and proteins are inserted independently into membranes.
Individual membrane lipids and proteins turn over independently and at different rates.
Topogenic sequences [eg, signal (amino terminal or internal) and stop-transfer] are important in determining the insertion and disposition of proteins in membranes.
Membrane proteins inside transport vesicles bud off the endoplasmic reticulum on their way to the Golgi; final sorting of many membrane proteins occurs in the trans-Golgi network.
Specific sorting sequences guide proteins to particular organelles such as lysosomes, peroxisomes, and mitochondria.
Various Disorders Result From Mutations in Genes Encoding Proteins Involved in Intracellular Transport
Some disorders reflecting abnormal peroxisomal function and abnormalities of protein synthesis in the ER and of the synthesis of lysosomal proteins have been listed earlier in this chapter (see Tables 49–4 and 49–7, respectively). Many other mutations affecting folding of proteins and their intracellular transport to various organelles have been reported, including neurodegenerative disorders such as Alzheimer disease Huntington disease and Parkinson disease. The elucidation of the causes of these various conformational disorders has contributed significantly to our understanding of molecular pathology. The term “diseases of proteostasis deficiency” has also been applied to diseases due to misfolding of proteins. Proteostasis is a composite word derived from protein homeostasis. Normal proteostasis is due to a balance of many factors, such as synthesis, folding, trafficking, aggregation, and normal degradation. If any one of these is disturbed (eg, by mutation, aging, cell stress, or injury), a variety of disorders can occur, depending on the particular proteins involved.
Potential therapies for the various diseases caused by protein dysfunction due to misfolding are aimed at correcting the conformational errors. One promising approach is to employ chaperones such as Hsp70 to promote proper folding. In addition, the antibiotic geldanamycin has been shown to activate heat shock proteins. Small drug molecules that act as chemical chaperones have also been shown prevent misfolding and restore protein function. These approaches, however, have so far been tested in animal experiments and in vitro systems and their effectiveness in humans remains to be established.
Many proteins are targeted to their destinations by signal sequences. A major sorting decision is made when proteins are partitioned between cytosolic (or free) and membrane-bound polyribosomes by virtue of the absence or presence of an N-terminal signal peptide.
Proteins synthesized on cytosolic polyribosomes are targeted by specific signal sequences to mitochondria, nuclei, peroxisomes, and the endoplasmic reticulum. Proteins which lack a signal remain in the cytosol.
Proteins synthesized on membrane bound polyribosomes initially enter the ER membrane or lumen, and many are ultimately destined for other membranes including the PM and that of the GA, for lysosomes and for secretion via exocytosis via transport from the ER → GA→ PM in transport vesicles.
Many glycosylation reactions occur in compartments of the Golgi, and proteins are further sorted in the trans-Golgi network.
Molecular chaperones stabilize unfolded or partially folded proteins. Chaperones are required for the correct targeting of proteins to their subcellular locations.
In posttranslational translocation, proteins are transported to their target organelles after their synthesis is complete. Proteins destined for mitochondria, the nucleus, and peroxisomes follow this route, as well as a minority of proteins targeted to the ER.
Most proteins enter the ER lumen by the cotranslational pathway, where translocation occurs during ongoing protein synthesis.
Proteins embedded in the ER membrane may inserted cotranslationally, posttranslationally or after transport to the GA (anterograde transport), transient retention and return to the ER (retrograde transport).
Harmful buildup of misfolded proteins triggers the unfolded protein response and they are degraded via the ERAD pathway. Proteins are tagged for degradation by the addition of a number of ubiquitin molecules and then enter the cytosol where they are broken down in proteasomes.
Different types of transport vesicles are coated with different proteins. Clathrin-coated vesicles are destined for exocytosis and lysosomes, while coat proteins I and II are associated with COPI and COPII vesicles, which are responsible retrograde and anterograde transport, respectively.
Transport vesicle processing is complex and requires many protein factors. Budding from the donor membrane is followed by movement through the cytosol, tethering, docking, and fusion with the target membrane.
Certain proteins (eg, precursors of albumin and insulin) are subjected to proteolysis while inside transport vesicles, producing the mature proteins.
Small GTPases (eg, Ran, Rab) and GEFs play key roles in many aspects of intracellular trafficking.
Vesicles formed from membranes of the ER and Golgi apparatus are asymmetrical in both lipid and protein content. The asymmetry is maintained during fusion of transport vesicles with the plasma membrane, so that the inside of the vesicles after fusion becomes the outside of the plasma membrane, and the cytoplasmic side of the vesicles remains facing the cytosol.
Asymmetry of both lipids and proteins is maintained during membrane assembly. Lipids and proteins are inserted independently and turn over at different rates. Details of the complex assembly process remain to be established.
Many disorders have been shown to be due to mutations in genes or to other factors that affect the folding of various proteins. These conditions have been referred to as conformational diseases, or alternatively as diseases of proteostatic deficiency. Promising therapeutic approaches include the use of chaperones such as Hsp70 and small molecules that can prevent misfolding and restore protein function.
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