chap 15

1

CHAPTER 15

INTRACELLULAR COMPARTMENTS

AND TRANSPORT

( 2004 Garland Science Publishing

Membrane-Enclosed Organelles

15-1Name the membrane-bounded compartments in a eucaryotic cell where each of the functions listed below takes place.

A.Photosynthesis

B.Transcription

C.Oxidative phosphorylation

D.Modification of secreted proteins

E.Steroid hormone synthesis

F.Degradation of worn-out organelles

G.New membrane synthesis

H.Breakdown of lipids and toxic molecules

15-2Label the structures of the cell indicated by the lines on the figure below:

Figure Q15-2

A.nucleus

B.free ribosomes

C.rough endoplasmic reticulum

D.Golgi apparatus

E.cytosol

F.endosome

G.plasma membrane

H.lysosome

I.mitochondrion

J.peroxisome

15-3You discover a fungus that contains a strange star-shaped organelle not found in any other eucaryotic cell you have seen. On further investigation you find the following

1.the organelle possesses a small genome in its interior.

2.the organelle is surrounded by two membranes.

3.vesicles do not pinch off the organelle membrane.

4.the interior of the organelle contains proteins similar to those of many bacteria.

5.the interior of the organelle contains ribosomes.

How might this organelle have arisen?

Protein Sorting

15-4For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once.

Plasma membrane proteins are inserted into the membrane in the __________________. The address information for protein sorting in a eucaryotic cell is contained in the __________________ of the proteins. Proteins enter the nucleus in their __________________ form. Proteins that remain in the cytosol do not contain a __________________. Proteins are transported into the Golgi apparatus via __________________. The proteins transported into the endoplasmic reticulum by __________________ are in their __________________ form.

amino acid sequenceGolgi apparatussorting signalendoplasmic reticulumplasma membranetransport vesiclesfoldedprotein translocatorsunfolded15-5What would happen in each of the following cases? Assume in each case that the protein involved is a soluble protein, not a membrane protein.

A.You add a signal sequence (for the ER) to the amino-terminal end of a normally cytosolic protein.

B.You change the hydrophobic amino acids in an ER signal sequence into charged amino acids.

C.You change the hydrophobic amino acids in an ER signal sequence into other, hydrophobic, amino acids.

D.You move the amino-terminal ER signal sequence to the carboxyl-terminal end of the protein.

15-6You are trying to identify the peroxisome-targeting sequence in the thiolase enzyme from yeast. The thiolase enzyme normally resides in the peroxisome and therefore must contain amino acid sequences that are used to target the enzyme for import into the peroxisome. To identify the targeting sequences, you create a set of hybrid genes that encode fusion proteins containing part of the thiolase protein fused to another protein, histidinol dehydrogenase (HDH). HDH is a cytosolic enzyme required for the synthesis of the amino acid histidine and cannot function if it is localized in the peroxisome. You genetically engineer a series of yeast cells to express these fusion proteins instead of their own versions of these enzymes. If the fusion proteins are imported into the peroxisome, the HDH portion of the protein cannot function and the yeast cells cannot grow on media lacking histidine. You obtain the following results:

Figure Q15-6

What region of the thiolase protein contains the peroxisomal targeting sequence? Explain your answer.

15-7What is the role of the nuclear localization sequence in a nuclear protein?

(a)It is bound by cytoplasmic proteins that direct the nuclear protein to the nuclear pore.

(b)It is a hydrophobic sequence that enables the protein to enter the nuclear membranes.

(c)It aids protein unfolding in order for the protein to thread through nuclear pores.

(d)It prevents the protein diffusing out of the nucleus via nuclear pores.

15-8A gene regulatory protein, A, contains a typical nuclear localization signal but surprisingly is usually found in the cytosol of cells. When the cell is exposed to hormones, protein A moves from the cytosol into the nucleus where it turns on genes involved in cell division. When you purify protein A from cells that have not been treated with hormones, you find that protein B is always complexed with it. To determine the function of protein B, you engineer cells lacking the gene for protein B. You compare normal and defective cells by using differential centrifugation to separate the nuclear fraction from the cytoplasmic fraction and then separate the proteins in these fractions by gel electrophoresis. You identify the presence of protein A and protein B by looking for their characteristic bands on the gel. The gel you run is shown below:

Figure Q15-8

On the basis of these results, what is the function of protein B? Explain your conclusion and propose a mechanism for how protein B works.

15-9Which of the following statements about import of proteins into mitochondria are TRUE?

(a)The signal sequences on mitochondrial proteins are usually carboxyl terminal.

(b)The first stage of import of a mitochondrial protein is across the outer membrane into the intermembrane space.

(c)Most mitochondrial proteins are not imported from the cytosol but are synthesized inside the mitochondria.

(d)Mitochondrial proteins are translocated across the inner and outer membranes simultaneously.

(e)Mitochondrial proteins cross the membrane in their native, folded state.

15-10Proteins destined to enter the endoplasmic reticulum

(a)are transported across the membrane after their synthesis is complete.

(b)are synthesized on free ribosomes in the cytosol.

(c)begin to cross the membrane while still being synthesized.

(d)cross the membrane in a folded state.

(e)all remain within the endoplasmic reticulum.

15-11After isolating the rough endoplasmic reticulum from the rest of the cytoplasm, you purify the RNAs attached to it. Which of the following proteins do you expect the RNA from the rough endoplasmic reticulum to encode?

(a)Soluble secreted proteins

(b)ER membrane proteins

(c)Mitochondrial membrane proteins

(d)Plasma membrane proteins

(e)Ribosomal proteins

15-12Briefly describe the mechanism by which the presence of an internal stop-transfer sequence in a protein causes the protein to become embedded in the lipid bilayer as a transmembrane protein with a single membrane-spanning region. Assume that the protein has an amino terminal signal sequence and just one internal hydrophobic stop-transfer sequence.

15-13Using genetic engineering techniques, you have created a set of proteins that contain two (and only two) conflicting signal sequences that specify different compartments. Predict which signal would win out for the following combinations. Explain your answers.

A.Signals for import into the nucleus and import into the ER.

B.Signals for export from the nucleus and import into the mitochondria.

C.Signals for import into mitochondria and retention in the ER.

15-14A protein traverses the plasma membrane three times in the orientation shown below (N=amino terminus, C=carboxyl terminus; the hydrophobic membrane-spanning regions are shown as open boxes). This protein is known to have a signal sequence that is cleaved by signal peptidase in the ER.

Figure Q15-14

Sketch the ER membrane and the arrangement of the newly synthesized protein chain after it has completed its entry into the ER membrane but before any action of signal peptidase. Be sure to label the cytosol, the ER lumen, the signal sequence, and the amino and carboxyl termini of the protein in your diagram.

15-15The figure below shows the orientation of a multipass transmembrane protein after it has completed its entry into the ER membrane (part A) and after it gets delivered to the plasma membrane (part B). This protein has an amino-terminal signal sequence (depicted as the dark grey membrane spanning box), which is cleaved off in the endoplasmic reticulum by signal peptidase. The other membrane-spanning domains in the protein are depicted as open boxes. Given that any hydrophobic membrane-spanning domain can act as either a start-transfer or a stop-transfer region, draw the final consequences of the actions described below on the orientation of the protein in the plasma membrane. Be sure to indicate on your drawing the extracellular space, the cytosolic face, and the plasma membrane, as well as the amino- and carboxyl-termini of the protein.

Figure Q15-15

A.Deleting the first signal sequence.

B.Changing the hydrophobic amino acids in the first, cleaved, sequence to charged amino acids.

C.Changing the hydrophobic residues in every other transmembrane sequence to charged residues, starting with the first, cleaved, signal sequence.

15-16Examine the multipass transmembrane protein shown in Figure Q15-16. 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.

Figure Q15-16

Vesicular Transport


Proteins are transported out of a cell via the __________________ or __________________ pathway. Fluids and macromolecules are transported into the cell via the __________________ pathway. All proteins being transported out of the cell pass through the __________________ and the __________________. Transport vesicles link organelles of the __________________ system. The formation of __________________ in the endoplasmic reticulum stabilizes protein structure.

carbohydrateGolgi apparatusdisulfide bondshydrogen bondsendocyticionic bondsendomembranelysosome

endoplasmic reticulumproteinendosomesecretoryexocytic

15-18Name two functions of the protein coat of vesicles that bud from membranous organelles used in vesicular transport.

15-19An individual transport vesicle

(a)contains only one type of protein in its lumen.

(b)will fuse with only one type of membrane.

(c)is endocytic if it is traveling toward the plasma membrane.

(d)is enclosed by a membrane with the same lipid and protein composition as the membrane of the donor organelle.

15-20In class we have discussed how v-SNAREs and t-SNARES mediate the recognition of a vesicle with its target membrane so that a vesicle displaying a particular type of v-SNARE will only fuse with a target membrane containing a complementary type of t-SNARE. It is also known that in some cases, v-SNAREs and t-SNAREs may also mediate fusion of identical membranes. In yeast cells, right before the formation of a new cell, vesicles derived from the vacuole will come together and fuse to form a new vacuole destined for the new cell. Unlike the situation weve discussed in class, the vacuolar vesicles contain both v-SNAREs and t-SNAREs. Your friend is trying to understand the role of these SNAREs in the formation of the new vacuole and wants to consult with you regarding the interpretation of his data.

Your friend has designed an ingenious assay for the fusion of vacuolar vesicles utilizing alkaline phosphatase. The protein alkaline phosphatase is made in a pro form that must be cleaved in order for the protein to be active. Your friend has designed two different strains of yeast: strain A produces the pro form of alkaline phosphatase (pro-Pase), while strain B produces the protease that can cleave pro-Pase into the active form (Pase). Neither strain has the active form of the alkaline phosphatase, but when vacuolar vesicles from the strains A and B are mixed, fusion of vesicles generates active alkaline phosphates, whose activity can be measured and quantified.

Figure Q15-20A

Your friend has taken each of these yeast strains and further engineered them so that they express only the v-SNAREs, the t-SNAREs, both (the normal situation), or neither SNARE. He then isolates vacuolar vesicles from all strains and tests the ability of each variant form of strain A to fuse with each variant form of strain B, using the alkaline phosphatase assay. The data are shown in the graph depicted in Figure Q15-20B. On this graph, the SNARE present on the vesicle of the particular yeast strain is indicated as v (for the presence of the v-SNARE) and t (for the presence of the t-SNARE).

Figure Q15-20 B

What does his data say about the requirements for v-SNAREs and t-SNAREs in the vacuolar vesicles? Be sure to comment on whether it is important to have a specific type of SNARE (that is, v- or t-SNARE) on each vesicle.

Secretory Pathway

15-21N-linked oligosaccharides on secreted glycoproteins are attached to

(a)nitrogen atoms in the polypeptide backbone.

(b)the serine or threonine in the sequence Asn-X-Ser/Thr.

(c)the amino terminus of the protein.

(d)the asparagine in the sequence Asn-X-Ser/Thr.

(e)the aspartic acid in the sequence Asp-X-Ser/Thr.

15-22Name two types of protein modification that can occur in the ER but not in the cytosol.

15-23If you were to remove the ER-retention signal from a protein that normally resides in the ER lumen, where do you expect the protein will ultimately end up? Be sure to explain your reasoning, for full credit.

15-24Match the set of labels below with the numbered label lines on Figure 15-24.

Figure Q15-24

A.Cisterna

B.Golgi stack

C.Secretory vesicle

D.trans Golgi network

E.cis Golgi network

15-25A plasma membrane protein carries an oligosaccharide containing mannose (Man), galactose (Gal), sialic acid (SA), and N-acetylglucosamine (GlcNAc). These sugars are added to the protein as it proceeds through the secretory pathway. First, a core oligosaccharide containing Man and GlcNAc is added, followed by Gal, Man, SA, and GlcNAc in a particular order. Each addition is catalyzed by a different transferase acting at a different stage as the protein proceeds through the secretory pathway. You have isolated mutants defective for each of the transferases, purified the membrane protein from each of the mutants, and identified which sugars are present in each mutant protein. The results are summarized in Table Q15-25.

Table Q15-25

Sugars present in the purified protein

Cell lacking:ManGalSA GlcNAc

A.Oligosaccharide

protein transferase

B.Galactose ++

transferase

C.SA transferase+++

D.GlcNAc +less than in

transferasenormal cells

From these results, match each of the transferases (A, B, C, D) to its subcellular location selected from the list below. (Assume that each location contains only one enzyme.)

1.Central Golgi cisternae

2.cis Golgi network

3.ER

4.trans Golgi network

15-26For each of the following sentences, choose one of the options enclosed in square brackets to make a correct statement.

New plasma membrane reaches the plasma membrane by the [regulated/constitutive] exocytosis pathway. New plasma membrane proteins reach the plasma membrane by the [regulated/constitutive] exocytosis pathway. Insulin is secreted from pancreatic cells by the [regulated/constitutive] exocytosis pathway. The interior of the trans Golgi network is [acidic/alkaline]. Proteins that are constitutively secreted [aggregate/do not aggregate] in the trans Golgi network.

15-27In a cell capable of regulated secretion, what are the three main classes of proteins that must be separated before they leave the trans Golgi network?

Endocytic Pathways

15-28Name three possible fates for an endocytosed molecule that has reached the endosome.


Eucaryotic cells are continually taking up materials from the extracellular space by the process of endocytosis. One type of endocytosis is __________________, which involves utilizing __________________ proteins to form small vesicles containing fluids and molecules. After these vesicles pinch off from the plasma membrane, they will fuse with the __________________, where the uptaken materials are sorted. A second type of endocytosis is __________________, which is used to take up large vesicles that can contain microorganisms and cellular debris. Macrophages are especially suited for this process, as they extend __________________ (sheetlike projections of their plasma membrane) to surround the invading microorganisms.

chaperoneGolgi apparatuspseudopodscholesterolmycobacteriumrough ERclathrinphagocytosisSNAREendosomepinocytosistranscytosis15-30Fibroblast cells from patients W, X, Y, and Z, who each have a different inherited defect, all contain inclusion bodies, which are lysosomes filled with undigested material. You wish to identify the cellular basis of these defects. The possibilities are:

1.a defect in one of the lysosomal hydrolases.

2.a defect in the phosphotransferase that is required for mannose-6-phosphate tagging of the lysosomal hydrolases.

3.a defect in the mannose-6-phosphate receptor, which binds mannose-6-phosphate tagged lysosomal proteins in the trans Golgi network and delivers them to lysosomes.

You find that when some of these mutant fibroblasts are incubated in media in which normal cells have been grown, the inclusion bodies disappear. This leads you to suspect that lysosomal hydrolases are being secreted by the constitutive exocytic pathway in normal cells and are being taken up by the mutant cells. (It is known that some mannose-6-phosphate receptor molecules are found in the plasma membrane and can take up and deliver lysosomal proteins via the endocytic pathway.) You incubate cells from each patient with media from normal cells and media from each of the other mutant cell cultures, and get the following results.

Media

FromFromFromFromFrom

normalculturesculturesculturescultures

cellsof W cellsof X cellsof Y cellsof Z cells

Cell Line

Normal+++++

W

X++

Y+++

Z+++

+ indicates that the cells appear normal; indicates that the cells still have inclusion bodies.

For each patient (W, X, Y, Z) indicate which of the defects (1, 2, 3) they are most likely to have.

15-31How is it that the low pH of lysosomes protects the rest of the cell from lysosomal enzymes in case the lysosome breaks?

How We Know: Tracking Protein and Vesicle Transport

15-32You have created a GFP fusion to a protein that is normally secreted from yeast cells. Since you have learned about the use of temperature-sensitive mutations in yeast to study protein and vesicle transport, you obtain a collection of three mutant yeast strains, each one defective in some aspect of the protein secretory process. Being a good scientist, you of course, also obtain a wild-type control strain. You decide to examine the fate of your GFP fusion protein in these various yeast strains and engineer the mutant strains to express your GFP fusion protein. However, in your excitement to do the experiment, you realize that you did not label any of the mutant yeast strains and no longer know which strain is defective in what process. You end up numbering your strains with the numbers 1 through 4, and then you carry out the experiment anyway, obtaining the following results (note that the black dots represent your GFP fusion protein):

Figure Q15-32

Name the process defective in each of these strains. Remember that one of these strains is your wild-type control.

Answers

15-1APhotosynthesis = chloroplast

B.Transcription = nucleus

C.Oxidative phosphorylation = mitochondrion

D.Modification of secreted proteins = Golgi apparatus and rough endoplasmic reticulum (ER)

E.Steroid hormone synthesis = smooth ER

F.Degradation of worn-out organelles = lysosome

G.New membrane synthesis = ER

H.Breakdown of lipids and toxic molecules = peroxisome

15-2 See Figure A15-2.

Figure A15-2

15-3A genome, a double membrane, ribosomes, and proteins similar to those found in bacteria are evidence for an organelle having evolved from an engulfed bacterium.

15-4Plasma membrane proteins are inserted into the membrane in the endoplasmic reticulum. The address information for protein sorting in a eucaryotic cell is contained in the amino acid sequence of the proteins. Proteins enter the nucleus in their folded form. Proteins that remain in the cytosol do not contain a sorting signal. Proteins are transported into the Golgi apparatus via transport vesicles. The proteins transported into the endoplasmic reticulum by protein translocators are in their unfolded form.15-5A.The protein will now be transported into the ER lumen.

B.The altered signal sequence will not be recognized and the protein will remain in the cytosol.

C.The protein will still be delivered into the ER. It is the distribution of hydrophobic amino acids that is important, not the actual sequence.

D.The protein will not enter the ER. Because the carboxyl terminus of the protein is the last part to be made, the ribosomes synthesizing this protein will not be recognized by the SRP and carried to the ER.

15-6The peroxisomal targeting sequence lies between amino acids number 100 and number 125. Any fusion protein containing this sequence can be targeted for import into the peroxisome (because the yeast cannot grow on media lacking histidine) while the fusion proteins lacking this region do not target the fusion protein for import into the peroxisome (because the yeast do grow on media lacking histidine). The most important pieces of data are the fusion proteins containing amino acids 100200 of the thiolase protein fused to HDH and the fusion protein containing amino acids 1125 of the thiolase protein fused to HDH. Both of these fusion proteins do not allow growth on media lacking histidine and can be used to define the minimal region necessary for targeting thiolase for import into the peroxisome.

(Note that although these experiments show that amino acids 100125 are necessary, these experiments do not show that this region is sufficient for peroxisomal targeting. It is possible that amino acids 100125 is sufficient, or, it could be that this region collaborates with redundant signals between amino acids 1100 or 125200.)

15-7(a)

15-8The data on the gel shows that protein A is always found in the nucleus in the absence of protein B. Therefore, any mechanism that is proposed must explain this result.

On possible answer is that protein B binds protein A and masks the nuclear localization signal. In the presence of hormone, protein B interacts with the hormone, which changes its conformation so that it can no longer bind protein A. When protein B no longer binds to protein A, the nuclear localization signal on protein A is now exposed and protein A can enter the nucleus. Therefore, in the absence of protein B, the nuclear localization signal on protein A is always exposed and protein A resides in the nucleus.

Another possible answer is that protein B binds protein A and sequesters it by keeping protein A in some subcellular compartment, away from the nucleus. In the presence of hormone, protein B interacts with the hormone, changing its conformation so that it can no longer bind to protein A. When protein B is not present, protein A can enter the nucleus in the presence or absence of hormone.

15-9(d)

15-10(c)

15-11(a), (b), and (d) The rough ER consists of ER membranes and polyribosomes that are in the process of translating and translocating proteins into the ER membrane and lumen. Thus all proteins that end up in the lysosome, Golgi apparatus, or plasma membrane, or are secreted, will be encoded by the RNAs associated with the rough ER. Mitochondrial and ribosomal proteins are translated on free cytosolic ribosomes.

15-12The amino-terminal signal sequence initiates translocation and the protein chain starts to thread through the translocation channel. When the stop-transfer sequence enters the translocation channel, the channel discharges both the signal sequence and the stop-transfer sequence sideways into the lipid bilayer. The signal sequence is then cleaved, so that the protein remains held in the membrane by the hydrophobic stop-transfer sequence.

15-13A.The protein would enter the ER. The signal for a protein to enter the ER is recognized as the protein is being synthesized and will end up either in the ER or on the ER membrane. Proteins destined for the nucleus get recognized by cytosolic nuclear transport proteins once they are fully synthesized and fully folded.

B.The protein would enter in the mitochondria. In order for a nuclear export signal to work, the protein would have to end up in the nucleus first and thus would need a nuclear import signal for the nuclear export signal to get utilized.

C.The protein would enter the mitochondria. In order to be retained in the ER, the protein needs to enter the ER. Since there is no signal for ER import, the ER retention signal would not function.

15-14The N-terminal signal sequence initiates translocation of the protein across the ER membrane. The signal sequence will be cleaved off by signal peptidase, leaving the amino-terminus of the protein in the luminal side of the ER membrane. Upon fusion to the plasma membrane, the amino terminus of the protein will reside in the extracellular space.

Figure A15-14

15-15A.Deleting the first signal sequence completely would convert the next membrane-spanning domain into an internal start-transfer signal and would invert the orientation of the protein (see Figure A15-15A).

B.Changing the hydrophobic amino acids to charged amino acids destroys the ability of the sequence both to act as a signal sequence and to become a membrane-spanning sequence. Therefore, the adjacent membrane spanning domain will now become an internal start-transfer sequence and the protein will be inverted, as seen above in part A. The mutated signal sequence would not get cleaved off, since it would remain on the cytoplasmic side of the membrane and signal peptidase is found only inside the ER (see Figure A15-15B).

C.Mutating every other membrane spanning region so that they are now charged (and thus cannot span the membrane) would decrease the number of transmembrane regions and increase the size of the internal loops between membrane-spanning regions (see Figure 15-15C).

Figure A15-15

15-16As shown in Figure A15-16, elimination of the first transmembrane segment (by making it hydrophilic) would be expected to reverse the orientation of the protein in the membrane. What originally was the second transmembrane segment (a stop-transfer signal), would now be read as a start-transfer signal and would have the opposite orientation in the membraneas would all the remaining transmembrane segments. Although the N-terminus would still be in the ER lumen, all the rest of the external parts of the protein would swap positions so that what was in the cytosol would now be in the ER lumen, and vice versa.

Figure A15-16

15-17Proteins are transported out of a cell via the secretory or exocytic pathway. Fluid and macromolecules are transported into the cell via the endocytic pathway. All proteins being transported out of the cell pass through the endoplasmic reticulum and the Golgi apparatus. Transport vesicles link organelles of the endomembrane system. The formation of disulfide bonds in the endoplasmic reticulum stabilizes protein structure.

15-181.The proteins in the coat help shape the membrane into a bud.

2.The proteins in the coat can also select cargoes for transport.

15-19Choice (b) is the correct answer. An individual vesicle may contain more than one type of protein in its lumen (choice (a)), all of which will contain the same sorting signal (or will lack specific sorting signals). Endocytic vesicles (choice (c)) generally move away from the plasma membrane. The vesicle membrane will not necessarily contain the same lipid and protein composition as the donor organelle, since the vesicle is formed from a selected subset of the organelle membrane from which it budded (choice (d)).

15-20In order to get maximal levels of vacuolar vesicle fusion, vesicles from each strain must carry both v-SNAREs and t-SNARES. Experiment 1, which represents the normal scenario, is the only experiment where 100% alkaline phosphatase activity is measured. However, as long as complementary SNAREs are present on the vesicles, some vesicle fusion does occur (see experiments 3, 4, 6, 7, 8, 9). If both vesicles are missing either v-SNAREs (experiment 2) or t-SNAREs (experiment 5) or both SNAREs (experiment 10 and 11), the level of fusion is very low. It does not matter whether a t- or v-SNARE is on the vesicle of a particular strain, as long as the vesicle from the other strain contains a complementary SNARE (compare experiments 3 and 4, 6 and 7, and 8 and 9).

15-21(d)

15-221.Proteins in the ER can undergo disulfide bond formation. (This does not occur in the cytosol because of its reducing environment.)

2.Proteins in the ER can undergo glycosylation. (Glycosylating enzymes are not found in the cytosol.)

(Signal-sequence cleavage is also an acceptable answer, although not really what this question is referring to.)

15-23The protein would end up in the extracellular space. Normally, the protein would go from the ER to the Golgi apparatus, get captured because of its ER-retrieval signal, and return to the ER. However, without the ER-retrieval signal, the protein would evade capture, ultimately leave the Golgi via the default pathway, and become secreted into the extracellular space. The protein would not be retained anywhere else along the secretory pathway, as it presumably has no signals to promote such localization since it normally resides in the ER lumen.

15-24A3; B1; C5; D4; E2

15-25A3 (oligosaccharide protein transferase = ER)

B1 (galactose transferase = central Golgi cisternae)

C4 (SA transferase = trans Golgi network)

D2 (GlcNAc transferase = cis Golgi network

Proteins are modified in a stepwise fashion in the Golgi apparatus, with early steps taking place in the cis Golgi, intermediate steps taking place in the central Golgi cisternae, and late steps occurring in the trans Golgi network. If each enzyme produces the substrate for the next step, then a mutant lacking the enzyme that catalyzes the addition of the first sugar will be missing all of the sugars, a mutant lacking the enzyme that catalyzes the addition of the second sugar will contain the first sugar but will lack the other three, and so on. By this logic, mannose and GlcNAc must be the first sugars added, additional GlcNAc the second, galactose the third, and SA the last. Hence, the oligosaccharide protein transferase must be in the ER, the GlcNAc transferase in the cis Golgi, the galactose transferase in the central Golgi, and the SA transferase in the trans Golgi.

15-26New plasma membrane reaches the plasma membrane by the constitutive exocytosis pathway. New plasma membrane proteins reach the plasma membrane by the constitutive exocytosis pathway. Insulin is secreted from pancreatic cells by the regulated exocytosis pathway. The interior of the trans Golgi network is acidic. Proteins that are constitutively secreted do not aggregate in the trans Golgi network.

15-27The three main classes of proteins that must be sorted before they leave the trans Golgi network in a cell capable of regulated secretion are (1) those destined for lysosomes, (2) those destined for secretory vesicles, and (3) those destined for immediate delivery to the cell surface.

15-281.recycled to the original membrane

2.destroyed in the lysosome

3.transcytosed across the cell to a different membrane.

15-29Eucaryotic cells are continually taking up materials from the extracellular space by the process of endocytosis. One type of endocytosis is pinocytosis, which involves utilizing clathrin proteins to form small vesicles containing fluids and molecules. After these vesicles pinch off from the plasma membrane, they will fuse with the endosome, where the uptaken materials are sorted. A second type of endocytosis is phagocytosis, which is used to take up large vesicles that can contain microorganisms and cellular debris. Macrophages are especially suited for this process, as they extend pseudopods (sheetlike projections of their plasma membrane) to surround the invading microorganisms.

15-30W3 (defect in mannose-6-phosphate receptor)

X2 (defect in phosphotransferase)

Y1; Z1 (defect in lysosomal hydrolases); these will be defects in two different lysosomal acid hydrolases.

A cell that has no mannose-6-phosphate receptor will be able to make all the lysosomal hydrolases properly, but will not be able to send them to the lysosome and will also not be able to scavenge hydrolases from the external media. Hence, this cell line cannot be rescued by culture media that has had lysosomal hydrolases secreted into it and thus will not be rescued by any of the media tested here. A cell line that has no phosphotransferase will be able to scavenge hydrolases from the external medium, but since all of the cells own hydrolases will lack the mannose-6-phosphate tag, it will be rescued only by media from a cell line that is able to make all of the hydrolases. Cell lines missing one hydrolase will be rescued by media from any cell line that is able to secrete that hydrolase in a mannose-6-phosphate tagged form; in addition, media from cultures of cells missing a hydrolase will rescue any cell line with another type of defect.

15-31The lysosomal enzymes are all acid hydrolases, which have optimal activity at the low pH (about 5.0) found in the interior of lysosomes. If a lysosome were to break, the acid hydrolases would find themselves at pH 7.2, the pH of the cytosol, and would therefore do little damage to cellular constituents.

15-32Strain A has protein accumulating in the ER, which means that this cell has a mutation that blocks transport from the ER to the Golgi apparatus. Strain B has secreted protein, and therefore is your wild-type control. Strain C has protein accumulating in the Golgi apparatus, and thus has a mutation that blocks exit of proteins from the Golgi apparatus. Strain D has protein accumulating in the cis-Golgi network, and thus has a mutation that blocks the travel of proteins through the Golgi cisternae.

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