THE_CELL_Molecular_Biology_of_Sixth_Edit-722-729.pdf

THE ENDOPLASMIC RETICULUM 687 How do misfolded proteins in the ER signal to the nucleus? There are three parallel pathways that execute the unfolded protein response (Figure 12–51A). The first pathway, which was initially discovered in yeast cells, is particularly remarkable. Misfolded proteins in...

THE ENDOPLASMIC RETICULUM 687 How do misfolded proteins in the ER signal to the nucleus? There are three parallel pathways that execute the unfolded protein response (Figure 12–51A). The first pathway, which was initially discovered in yeast cells, is particularly remarkable. Misfolded proteins in the ER activate a transmembrane protein kinase in the ER, called IRE1, which causes the kinase to oligomerize and phosphorylate itself. (Some cell-surface receptor kinases in the plasma membrane are activated in a three sensors for misfolded proteins (A) IRE1 PERK ER membrane ER LUMEN CYTOSOL P P P Figure 12–51 The unfolded protein response. (A) By three parallel intracellular signaling pathways, the accumulation of misfolded proteins in the ER lumen signals to the nucleus to activate the transcription of genes that encode proteins that help the cell cope with misfolded proteins in the ER. (B) Regulated RNA splicing is a key regulatory switch in pathway 1 of the unfolded protein response (Movie 12.6). ATF6 P kinase domain ribonuclease domain PHOSPHORYLATION INACTIVATES TRANSLATION INITIATION FACTOR REDUCTION OF PROTEINS ENTERING THE ER REGULATED mRNA SPLICING INITIATES TRANSLATION OF SELECTIVE TRANSLATION OF REGULATED PROTEOLYSIS IN GOLGI APPARATUS RELEASES TRANSCRIPTION REGULATORY PROTEIN 1 TRANSCRIPTION REGULATORY PROTEIN 2 TRANSCRIPTION REGULATORY PROTEIN 3 ACTIVATION OF GENES TO INCREASE PROTEIN-FOLDING CAPACITY OF ER (B) 1 2 3 4 MISFOLDED PROTEINS IN ER SIGNAL THE NEED FOR MORE ER CHAPERONES. THEY BIND TO AND ACTIVATE A TRANSMEMBRANE KINASE misfolded proteins 1 misfolded protein bound to chaperone 2 ENDORIBONUCLEASE CUTS SPECIFIC RNA MOLECULES AT TWO POSITIONS, REMOVING AN INTRON TWO EXONS ARE LIGATED TO FORM AN ACTIVE mRNA kinase domain P 7 P transmembrane protein kinase (sensor) 3 ribonuclease domain intron pre-mRNA CYTOSOL 4 5 mRNA IS TRANSLATED TO MAKE A TRANSCRIPTION REGULATOR 6 TRANSCRIPTION REGULATOR ENTERS NUCLEUS AND ACTIVATES GENES ENCODING ER CHAPERONES ER LUMEN ER chaperone ACTIVATED KINASE UNMASKS AN ENDORIBONUCLEASE ACTIVITY mRNA exon exon 5 7 transcription regulator nuclear pore 6 CHAPERONES ARE MADE IN ER, WHERE THEY HELP FOLD PROTEINS chaperone gene chaperone mRNA NUCLEUS 688 Chapter 12: Intracellular Compartments and Protein Sorting similar way, as discussed in Chapter 15.) The oligomerization and autophosphorylation of IRE1 activates an endoribonuclease domain in the cytosolic portion of the same molecule, which cleaves a specific cytosolic mRNA molecule at two positions, excising an intron. (This is a unique exception to the rule that introns are spliced out while the RNA is still in the nucleus.) The separated exons are then joined by an RNA ligase, generating a spliced mRNA, which is translated to produce an active transcription regulatory protein. This protein activates the transcription of genes encoding the proteins that help mediate the unfolded protein response (Figure 12–51B). Misfolded proteins also activate a second transmembrane kinase in the ER, PERK, which inhibits a translation initiation factor by phosphorylating it, thereby reducing the production of new proteins throughout the cell. One consequence of the reduction in protein synthesis is to reduce the flux of proteins into the ER, thereby reducing the load of proteins that need to be folded there. Some proteins, however, are preferentially translated when translation initiation factors are scarce (discussed in Chapter 7, p. 424), and one of these is a transcription regulator that helps activate the transcription of the genes encoding proteins active in the unfolded protein response. Finally, a third transcription regulator, ATF6, is initially synthesized as a transmembrane ER protein. Because it is embedded in the ER membrane, it cannot activate the transcription of genes in the nucleus. When misfolded proteins accumulate in the ER, however, the ATF6 protein is transported to the Golgi apparatus, where it encounters proteases that cleave off its cytosolic domain, which can now migrate to the nucleus and help activate the transcription of genes encoding proteins involved in the unfolded protein response. (This mechanism is similar to that described in Figure 12–16 for activation of the transcription regulator that controls cholesterol biosynthesis.) The relative importance of each of these three pathways in the unfolded protein response differs in different cell types, enabling each cell type to tailor the unfolded protein response to its particular needs. Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor As discussed in Chapter 10, several cytosolic enzymes catalyze the covalent addition of a single fatty acid chain or prenyl group to selected proteins. The attached lipids help direct and attach these proteins to cell membranes. A related process is catalyzed by ER enzymes that covalently attach a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of some membrane proteins destined for the plasma membrane. This linkage forms in the lumen of the ER, where, at the same time, the transmembrane segment of the protein is cleaved off (Figure 12–52). A large number of plasma membrane proteins are modified in this way. Since they are attached to the exterior of the plasma membrane only by their GPI anchors, CYTOSOL COOH ER LUMEN glycosylphosphatidylinositol P H2N P cleaved C-terminal peptide COOH NH2 P P inositol ethanolamine protein bound to membrane by GPI anchor NH2 NH2 Figure 12–52 The attachment of a GPI anchor to a protein in the ER. GPIanchored proteins are targeted to the ER membrane by an N-terminal signal sequence (not shown), which is removed (see Figure 12–42). Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence of 15–20 amino acids; the rest of the protein is in the ER lumen. Within less than a minute, an enzyme in the ER cuts the protein free from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled GPI intermediate. The sugar chain contains an inositol attached to the lipid from which the GPI anchor derives its name. It is followed by a glucosamine and three mannoses. The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the ER membrane; if this signal is added to other proteins, they too become modified in this way. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the ER and eventually on the exterior of the plasma membrane. THE ENDOPLASMIC RETICULUM OH P OH 2 CoA 2 C O C O C O C O fatty acid fatty acid fatty acid OH CoA fatty acid CYTOSOL OH CoA P P 3 CH2 CH O O C O C O fatty acid 1 P phosphatidic acid CH2 Pi 4 choline CMP P C choline phosphotransferase phosphatase acyl transferase acyl-CoA ligase lipid bilayer of ER C OH CH2 CH O O C O C O diacylglycerol CH2 choline P 5 CH2 CH O O CH2 C O C O fatty acid OH CDP-choline CH2 fatty acid 2 CoA CH fatty acid fatty acid binding protein CH2 fatty acid O glycerol 3-phosphate fatty acid C cid ty a fat 689 phosphatidylcholine ER LUMEN they can in principle be released from cells in soluble form in response to signals that activate a specific phospholipase in the plasma membrane. Trypanosome parasites, for example, use this mechanism to shed their coat of GPI-anchored surface proteins when attacked by the immune system. GPI anchors may also be used to direct plasma membrane proteins into lipid rafts and thus segregate the proteins from other membrane proteins (see Figure 10–13). The ER Assembles Most Lipid Bilayers m12.57/12.56 The ER membrane is the site of synthesis of nearly MBoC6 all of the cell’s major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The major phospholipid made is phosphatidylcholine, which can be formed in three steps from choline, two fatty acids, and glycerol phosphate (Figure 12–53). Each step is catalyzed by enzymes in the ER membrane, which have their active sites facing the cytosol, where all of the required metabolites are found. Thus, phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane. Because fatty acids are not soluble in water, they are shepherded from their sites of synthesis to the ER by a fatty acid binding protein in the cytosol. After arrival in the ER membrane and activation with CoA, acyl transferases successively add two fatty acids to glycerol phosphate to produce phosphatidic acid. Phosphatidic acid is sufficiently water-insoluble to remain in the lipid bilayer; it cannot be extracted from the bilayer by the fatty acid binding proteins. It is therefore this first step that enlarges the ER lipid bilayer. The later steps determine the head group of a newly formed lipid molecule and therefore the chemical nature of the bilayer, but they do not result in net membrane growth. The two other major membrane phospholipids—phosphatidylethanolamine and phosphatidylserine (see Figure 10–3)—as well as the minor phospholipid phosphatidylinositol (PI), are all synthesized in this way. Because phospholipid synthesis takes place in the cytosolic leaflet of the ER lipid bilayer, there needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer. In synthetic lipid bilayers, lipids do not “flip-flop” in this way (see Figure 10–10). In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flipflop.” This rapid trans-bilayer movement is mediated by a poorly characterized Figure 12–53 The synthesis of phosphatidylcholine. As illustrated, this phospholipid is synthesized from glycerol 3-phosphate, cytidine-diphosphocholine (CDP-choline), and fatty acids delivered to the ER by a cytosolic fatty acid binding protein. 690 Chapter 12: Intracellular Compartments and Protein Sorting (A) ER MEMBRANE (B) PLASMA MEMBRANE CYTOSOL CELL EXTERIOR asymmetric lipid bilayer of plasma membrane lipid bilayer of endoplasmic reticulum ER LUMEN CYTOSOL PHOSPHOLIPID SYNTHESIS ADDS TO CYTOSOLIC HALF OF THE BILAYER DELIVERY OF NEW MEMBRANE BY EXOCYTOSIS asymmetric growth of bilayer SCRAMBLASE CATALYZES FLIPPING OF PHOSPHOLIPID MOLECULES FROM CYTOSOLIC TO LUMENAL LEAFLET FLIPPASE CATALYZES FLIPPING OF SPECIFIC PHOSPHOLIPIDS TO CYTOSOLIC MONOLAYER Figure 12–54 The role of phospholipid translocators in lipid bilayer synthesis. (A) Because new lipid molecules are added only to the cytosolic half of the ER membrane bilayer and lipid molecules do not flip spontaneously from one monolayer to the other, a transmembrane phospholipid translocator (called a scramblase) is required to transfer lipid molecules from the cytosolic half to the lumenal half so that the membrane grows as a bilayer. The scramblase is not specific for particular phospholipid head groups and therefore equilibrates the different phospholipids between the two monolayers. (B) Fueled by ATP hydrolysis, a head-group-specific flippase in the plasma membrane actively flips phosphatidylserine and phosphatidylethanolamine directionally from the extracellular to the cytosolic leaflet, creating the characteristically asymmetric lipid bilayer of the plasma membrane of animal cells (see Figure 10–15). symmetric growth of both halves of bilayer phospholipid translocator called a scramblase, which nonselectively equilibrates phospholipids between the two leaflets of the lipid bilayer (Figure 12–54). Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane. The plasma membrane contains a different type of phospholipid translocator that belongs to the family of P-type pumps (discussed in Chapter 11). These flipMBoC6 m12.58/12.57 pases specifically recognize those phospholipids that contain free amino groups in their head groups (phosphatidylserine and phosphatidylethanolamine—see Figure 10–3) and transfers them from the extracellular to the cytosolic leaflet, using the energy of ATP hydrolysis. The plasma membrane therefore has a highly asymmetric phospholipid composition, which is actively maintained by the flippases (see Figure 10–15). The plasma membrane also contains a scramblase but, in contrast to the ER scramblase, which is always active, the plasma membrane enzyme is regulated and only activated in some situations, such as in apoptosis and in activated platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and degrade the dead cell. The ER also produces cholesterol and ceramide (Figure 12–55). Ceramide is made by condensing the amino acid serine with a fatty acid to form the amino alcohol sphingosine (see Figure 10–3); a second fatty acid is then covalently added to form ceramide. The ceramide is exported to the Golgi apparatus, where it serves as a precursor for the synthesis of two types of lipids: oligosaccharide chains are added to form glycosphingolipids (glycolipids; see Figure 10–16), and phosphocholine head groups are transferred from phosphatidylcholine to other ceramide molecules to form sphingomyelin (discussed in Chapter 10). Thus, both glycolipids and sphingomyelin are produced relatively late in the process of membrane synthesis. Because they are produced by enzymes that have their active sites exposed to the Golgi lumen, they are found exclusively in the noncytosolic leaflet of the lipid bilayers that contain them. OH OH CH CH CH NH CH C (CH2)12 (CH2)16 CH3 CH3 CH2 O CERAMIDE Figure 12–55 The structure of ceramide. MBoC6 m12.59/12.58 THE ENDOPLASMIC RETICULUM 691 As discussed in Chapter 13, the plasma membrane and the membranes of the Golgi apparatus, lysosomes, and endosomes all form part of a membrane system that communicates with the ER by means of transport vesicles, which transfer both proteins and lipids. Mitochondria and plastids, however, do not belong to this system, and they therefore require different mechanisms to import proteins and lipids for growth. We have already seen that they import most of their proteins from the cytosol. Although mitochondria modify some of the lipids they import, they do not synthesize lipids de novo; instead, their lipids have to be imported from the ER, either directly or indirectly by way of other cell membranes. In either case, special mechanisms are required for the transfer. The details of how lipid distribution between different membranes is catalyzed and regulated are not known. Water-soluble carrier proteins called phospholipid exchange proteins (or phospholipid transfer proteins) are thought to transfer individual phospholipid molecules between membranes, functioning much like fatty acid binding proteins that shepherd fatty acids through the cytosol (see Figure 12–54). In addition, mitochondria are often seen in close juxtaposition to ER membranes in electron micrographs, and specific junction complexes have been identified that hold the ER and outer mitochondrial membrane in close proximity. It is thought that these junction complexes provide specific contact-dependent lipid transfer mechanisms that operate between these adjacent membranes. Summary The extensive ER network serves as a factory for the production of almost all of the cell’s lipids. In addition, a major portion of the cell’s protein synthesis occurs on the cytosolic surface of the rough ER: virtually all proteins destined for secretion or for the ER itself, the Golgi apparatus, the lysosomes, the endosomes, and the plasma membrane are first imported into the ER from the cytosol. In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and N-linked oligosaccharides are added. The pattern of N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, polyubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to help the ER cope. Only proteins that carry a special ER signal sequence are imported into the ER. The signal sequence is recognized by a signal-recognition particle (SRP), which binds both the growing polypeptide chain and the ribosome and directs them to a receptor protein on the cytosolic surface of the rough ER membrane. This binding to the ER membrane initiates the translocation process that threads a loop of polypeptide chain across the ER membrane through the hydrophilic pore of a protein translocator. Soluble proteins—destined for the ER lumen, for secretion, or for transfer to the lumen of other organelles—pass completely into the ER lumen. Transmembrane proteins destined for the ER or for other cell membranes are translocated part way across the ER membrane and remain anchored there by one or more membrane-spanning α-helical segments in their polypeptide chains. These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during the translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein. The asymmetry of protein insertion and glycosylation in the ER establishes the sidedness of the membranes of all the other organelles that the ER supplies with membrane proteins. What we don’t know • How do nuclear import receptors negotiate the tangled gel-like interior of a nuclear pore complex so efficiently? • Is the nuclear pore complex a rigid structure or can it expand and contract, depending on the cargo transported? • Sequence comparisons show that signal sequences for an individual protein such as insulin are quite conserved across species, much more so than would be expected from our current understanding that all that matters for their function are general structural features such as hydrophobicity. What other functions might signal sequences have that could account for their evolutionary sequence conservation? • How are polyribosomes on the endoplasmic reticulum membrane arranged so that the next initiating ribosome will find an unoccupied translocator? • Why does the signal-recognition particle have an indispensable RNA subunit? 692 Chapter 12: Intracellular Compartments and Protein Sorting Problems Which statements are true? Explain why or why not. 12–1 Like the lumen of the ER, the interior of the nucleus is topologically equivalent to the outside of the cell. 12–2 ER-bound and free ribosomes, which are structurally and functionally identical, differ only in the proteins they happen to be making at a particular time. 12–3 To avoid the inevitable collisions that would occur if two-way traffic through a single pore were allowed, nuclear pore complexes are specialized so that some mediate import while others mediate export. nucleoplasmin preparation intact one tail nuclear injection cytoplasmic injection Figure Q12–1 Cellular location of injected nucleoplasmin and nucleoplasmin components (Problem 12–7). Schematic diagrams of autoradiographs show the cytoplasm and nucleus with the location of nucleoplasmin indicated by the red areas. heads only 12–4 Peroxisomes are found in only a few specialized types of eukaryotic cell. Discuss the following problems. 12–5 tails only What is the fate of a protein with no sorting signal? 12–6 The rough ER is the site of synthesis of many classes of membrane proteins. Some of these proteins remain in the ER, whereas others are sorted to compartments such as the Golgi apparatus, lysosomes, and the plasma membrane. One measure of the difficulty of the sorting problem is the degree of “purification” that must be achieved during transport from the ER. Are proteins bound for the plasma membrane common or rare among all ER membrane proteins? A few simple considerations allow one to answer this question. In a typical growing cell that is dividing once every 24 hours, the equivalent of one new plasma membrane must transit the ER every day. If the ER membrane is 20 times the area of a plasma membrane, what is the ratio of plasma membrane proteins to other membrane proteins in the ER? (Assume that all proteins on their way to the plasma membrane remain in the ER for 30 minutes on average before exiting, and that the ratio of proteins to lipids in the ER and plasma membranes is the same.) 12–7 Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to residents of the nucleus such as chromosomes, or whether they were actively imported and accumulated regardless of their affinity for nuclear components. A classic experiment that addressed this problem used several forms of radioactive nucleoplasmin, which is a large pentameric protein involved in chromatin assembly. In this experiment, either the intact protein or the nucleoplasmin heads, tails, or heads with a single tail were injected into the cytoplasm of a frog oocyte or into the nucleus (Figure Q12–1). All forms of nucleoplasmin, except heads, accumulated in the nucleus when injected into the cytoplasm, and all forms were retained in the nucleus when injected there. A. What portion of the nucleoplasmin molecule is responsible for localization in the nucleus? B. How do these experiments distinguish between active transport, in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion, in which a binding site for a nuclear component allows accumulation in the nucleus? 12–8 Assuming that 32 million histone octamers are required to package the human genome, how many histone molecules must be transported per second per nuclear pore complex in cells whose nuclei contain 3000 nuclear pores and are dividing once per day? 12–9 The nuclear pore complex (NPC) creates a barrier to the free exchange of molecules between the nucleus and cytosol, but in a way that remains mysterious. In yeast, for Problems p12.04/12.05 example, the central pore of the NPC has a diameter of 35 nm and is 30 nm long, which is somewhat smaller than its vertebrate counterpart. Even so, it is large enough to accommodate virtually all components of the cytosol. Yet the pore allows passive diffusion of molecules only up to about 40 kd; entry of anything larger requires help from a nuclear import receptor. Selective permeability is controlled by protein components of the NPC that have unstructured, polar tails extending into the central pore. These tails are characterized by periodic repeats of the hydrophobic amino acids phenylalanine (F) and glycine (G). At high enough concentration (~50 mM), the FG-repeat domains of these proteins can form a gel, with a meshwork of interactions between the hydrophobic FG repeats (Figure Q12–2A). These gels allow passive diffusion of small molecules, but they prevent entry of larger proteins such as the fluorescent protein mCherry fused to maltose binding protein (MBP) (Figure Q12–2B). (The fusion to MBP makes mCherry too large to enter the nucleus by passive diffusion.) However, if the nuclear import receptor, importin, is fused to a similar protein, MBP-GFP, the importin-MBP-GFP fusion readily enters the gel (Figure Q12–2B). CHAPTER 12 END-OF-CHAPTER PROBLEMS (A) (B) solution gel 30 s 10 min 30 min MBP-mCherry 30 s 10 min 30 min importin-MBP-GFP Figure Q12–2 FG-repeat gel and influx of proteins into the nucleus (Problem 12–9). (A) Cartoon of the meshwork (gel) formed by pairwise interactions between hydrophobic FG repeats. For FG-repeats separated by 17 amino acids, as is typical, the mesh formed by extended amino acid side chains would correspond to about 4 nm on a side, which would be large enough to account for the characteristic passive diffusion of proteins through nuclear pores. (B) Diffusion of MBP-mCherry and importin-MBP-GFP into a gel of FG-repeats. In each group, the solution is shown at left and the gel at right. The bright areas indicate regions that contain the fluorescent proteins. Problems p12.201/12.04 A. FG-repeats only form gels in vitro at relatively high concentration (50 mM). Is this concentration reasonable for FG repeats in the NPC core? In yeast, there are about 5000 FG-repeats in each NPC. Given the dimensions of the yeast nuclear pore (35 nm diameter and 30 nm length), calculate the concentration of FG-repeats in the cylindrical volume of the pore. Is this concentration comparable to the one used in vitro? B. A second question is whether the diffusion of importin-MBP-GFP through the FG-repeat gel is fast enough to account for the efficient flow of materials between the nucleus and cytosol. From experiments of the type shown in Figure Q12–2B, the diffusion coefficient (D) of importin-MBP-GFP through the FG-repeat gel was determined to be about 0.1 μm2/s. The equation for diffusion is t = x2/2D, where t is time and x is distance. Calculate the time it would take importin-MBP-GFP to diffuse through a yeast nuclear pore (30 nm) if the pore consisted of a gel of FG-repeats. Does this time seem fast enough for the needs of a eukaryotic cell? 12–10 Components of the TIM complexes, the multisubunit protein translocators in the mitochondrial inner membrane, are much less abundant than those of the TOM 693 complex. They were initially identified using a genetic trick. The yeast Ura3 gene, whose product is an enzyme that is normally located in the cytosol where it is essential for synthesis of uracil, was modified so that the protein carried an import signal for the mitochondrial matrix. A population of cells carrying the modified Ura3 gene in place of the normal gene was then grown in the absence of uracil. Most cells died, but the rare cells that grew were shown to be defective for mitochondrial import. Explain how this selection identifies cells with defects in components required for import into the mitochondrial matrix. Why don’t normal cells with the modified Ura3 gene grow in the absence of uracil? Why do cells that are defective for mitochondrial import grow in the absence of uracil? 12–11 If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import? 12–12 Why do mitochondria need a special translocator to import proteins across the outer membrane, when the membrane already has large pores formed by porins? 12–13 Examine the multipass transmembrane protein shown in Figure Q12–3. What would you predict would be the effect of converting the first hydrophobic transmembrane segment to a hydrophilic segment? Sketch the arrangement of the modified protein in the ER membrane. COOH 1 3 5 CYTOSOL ER LUMEN 2 4 6 NH2 Figure Q12–3 Arrangement of a multipass transmembrane protein in the ER membrane (Problem 12–13). Blue hexagons represent covalently attached oligosaccharides. 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