CHAPTER 15

INTRACELLULAR COMPARTMENTS
AND TRANSPORT
2004 Garland Science Publishing

Membrane-Enclosed Organelles

15-1 Name 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

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15-2 Label 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

15-3 You 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?

248
Protein Sorting

15-4 For 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 sequence Golgi apparatus sorting signal

endoplasmic reticulum plasma membrane transport vesicles
folded protein translocators unfolded

15-5 What 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.

249
15-6 You 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.

250
15-7 What 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.

15-8 A 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.

15-9 Which 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.

251
15-10 Proteins 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.

15-11 After 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

15-12 Briefly 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.

15-13 Using 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.

15-14 A 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.

252
15-15 The 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.
15-16 Examine 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

253
Vesicular Transport

15-17 For 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.

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.

carbohydrate Golgi apparatus

disulfide bonds hydrogen bonds
endocytic ionic bonds
endomembrane lysosome
endoplasmic reticulum protein
endosome secretory
exocytic

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

15-19 An 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.

254
15-20 In 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).

255
 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

15-21 N-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.

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

15-23 If 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.

256
15-24 Match 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

257
15-25 A 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: Man Gal SA GlcNAc
A. Oligosaccharide
protein transferase
B. Galactose + +
transferase
C. SA transferase + + +
D. GlcNAc + less than in
transferase normal 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

15-26 For 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.

15-27 In 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?

258
Endocytic Pathways

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

15-29 For 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.

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.

chaperone Golgi apparatus pseudopods

cholesterol mycobacterium rough ER
clathrin phagocytosis SNARE
endosome pinocytosis transcytosis

259
15-30 Fibroblast 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
From From From From From
normal cultures cultures cultures cultures
cells of W cells of X cells of Y cells of 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.

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

260
How We Know: Tracking Protein and Vesicle Transport

15-32 You 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.

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Answers

15-1 A Photosynthesis = 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

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

15-4 Plasma 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.

262
15-5 A. 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.

15-6 The 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)

15-8 The 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)

263
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.

15-12 The 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.

15-13 A. 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.

15-14 The 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

264
15-15 A. 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

15-16 As 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

265
15-17 Proteins 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.

15-18 1. The proteins in the coat help shape the membrane into a bud.
2. The proteins in the coat can also select cargoes for transport.

15-19 Choice (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)).

15-20 In 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)

15-22 1. 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.)

15-23 The 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.

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

266
15-25 A3 (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.

15-26 New 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.

15-27 The 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.

15-28 1. recycled to the original membrane

2. destroyed in the lysosome
3. transcytosed across the cell to a different membrane.

15-29 Eucaryotic 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.

267
15-30 W3 (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.

15-31 The 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.

15-32 Strain 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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