CHAPTER 15
INTRACELLULAR COMPARTMENTS AND
TRANSPORT
 2009 Garland Science Publishing
Membrane-Enclosed Organelles
15-1
Which of the following statements about the endoplasmic reticulum (ER) is false?
(a)
The ER is the major site for new membrane synthesis in the cell.
(b)
Proteins to be delivered to the ER lumen are synthesized on smooth ER.
(c)
Steroid hormones are synthesized on the smooth ER.
(d)
The ER membrane is contiguous with the outer nuclear membrane.
15-2
Which of the following statements about membrane-enclosed organelles is true?
(a)
In a typical cell, the area of the endoplasmic reticulum membrane far
exceeds the area of plasma membrane.
(b)
The nucleus is the only organelle that is surrounded by a double
membrane.
(c)
Other than the nucleus, most organelles are small and thus, in a typical
cell, only about 10% of a cell’s volume is occupied by membraneenclosed organelles; the other 90% of the cell volume is the cytosol.
(d)
The nucleus is the only organelle that contains DNA.
15-3
Name the membrane-enclosed 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
15-4
Label the structures of the cell indicated by the lines in Figure Q15-4:
Figure Q15-4
A.
B.
C.
D.
E.
F.
G.
H.
I.
nucleus
free ribosomes
rough endoplasmic reticulum
Golgi apparatus
cytosol
endosome
plasma membrane
lysosome
mitochondrion
15-5
Which of the following organelles are not part of the endomembrane system?
(a)
Golgi apparatus
(b)
the nucleus
(c)
mitochondria
(d)
lysosomes
15-6
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 from 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
15-7
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; use each word
or phrase 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
endoplasmic reticulum
folded
Golgi apparatus
plasma membrane
protein translocators
sorting signal
transport vesicles
unfolded
15-8
Where are proteins in the chloroplast synthesized?
(a)
in the cytosol
(b)
in the chloroplast
(c)
on the endoplasmic reticulum
(d)
in both the cytosol and the chloroplast
15-9
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 N-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 N-terminal ER signal sequence to the C-terminal end of the
protein.
15-10 Which of the following statements is true?
(a)
The signal sequences on mitochondrial proteins are usually C-terminal.
(b)
Most mitochondrial proteins are not imported from the cytosol but are
synthesized inside the mitochondria.
(c)
Chaperone proteins in the mitochondria facilitate the movement of
proteins across the outer and inner mitochondrial membranes.
(d)
Mitochondrial proteins cross the membrane in their native, folded state.
15-11 Most 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)
(d)
begin to cross the membrane while still being synthesized.
remain within the endoplasmic reticulum.
15-12 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)
plasma membrane proteins
(d)
all of the above
15-13 In which cellular location would you expect to find ribosomes translating mRNAs
that encode ribosomal proteins?
(a)
the nucleus
(b)
on the rough ER
(c)
in the cytosol
(d)
in the lumen of the ER
15-14 You are trying to identify the peroxisome-targeting sequence in the thiolase
enzyme in 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 a
medium lacking histidine. You obtain the results shown in Figure Q15-14.
Figure Q15-14
What region of the thiolase protein contains the peroxisomal targeting sequence?
Explain your answer.
15-15 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 in protein unfolding so that the protein can thread through nuclear
pores.
(d)
It prevents the protein from diffusing out of the nucleus through nuclear
pores.
15-16 A gene regulatory protein, A, contains a typical nuclear localization signal but
surprisingly is usually found in the cytosol. 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 in Figure Q15-16.
Figure Q15-16
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.
15-17 Your friend works in a biotechnology company and has discovered a drug that
blocks the ability of Ran to exchange GDP for GTP. What is the most likely effect
of this drug on nuclear transport?
(a)
Nuclear transport receptors would be unable to bind cargo.
(b)
Nuclear transport receptors would be unable to enter the nucleus.
(c)
(d)
Nuclear transport receptors would be unable to release their cargo in the
nucleus.
Nuclear transport receptors would interact irreversibly with the nuclear
pore fibrils.
15-18 You are interested in Fuzzy, a soluble protein that functions within the ER lumen.
Given that information, which of the following statement must be true?
(a)
Fuzzy has a C-terminal signal sequence that binds to SRP.
(b)
Only one ribosome can be bound to the mRNA encoding Fuzzy during
translation.
(c)
Fuzzy must contain a hydrophobic stop-transfer sequence.
(d)
Once the signal sequence from Fuzzy has been cleaved, the signal peptide
will be ejected into the ER membrane and degraded.
15-19 Figure Q15-19 shows the organization of a protein that normally resides in the
plasma membrane. The boxes labeled 1 and 2 represent membrane-spanning
sequences and the arrow represents a site of action of signal peptidase. Given this
diagram, which of the following statements must be true?
Figure Q15-19
(a)
(b)
(c)
(d)
The N-terminus of this protein is cytoplasmic.
The C-terminus of this protein is cytoplasmic.
The mature version of this protein will span the membrane twice, with
both the N and C-termini in the cytoplasm.
None of the above.
15-20 Briefly describe the mechanism by which 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 N-terminal signal sequence and just one internal hydrophobic stoptransfer sequence.
15-21 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.
15-22 Figure Q15-22 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 N-terminal signal sequence
(depicted as the dark gray membrane-spanning box), which signal peptidase
cleaves off in the endoplasmic reticulum. The other membrane-spanning domains
in the protein are represented as open boxes. Given that any hydrophobic
membrane-spanning domain can act as either a start-transfer region or a stoptransfer region, draw the final consequences of the actions described below on the
orientation of the protein in the plasma membrane. Indicate on your drawing the
extracellular space, the cytosolic face, and the plasma membrane, as well as the
N- and C-termini of the protein.
Figure Q15-22
A.
B.
C.
deleting the first signal sequence
changing the hydrophobic amino acids in the first, cleaved, sequence to
charged amino acids
changing the hydrophobic residues in every other transmembrane
sequence to charged residues, starting with the first, cleaved, signal
sequence
Vesicular Transport
15-23 Which of the following choices reflects the appropriate order through which a
protein destined for the plasma membrane travels?
(a)
lysosome → endosome → plasma membrane
(b)
ER → lysosome → plasma membrane
(c)
Golgi → lysosome → plasma membrane
(d)
ER → Golgi → plasma membrane
15-24 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; use each word
or phrase 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
disulfide bonds
endocytic
endomembrane
endoplasmic reticulum
endosome
exocytic
Golgi apparatus
hydrogen bonds
ionic bonds
lysosome
protein
secretory
15-25 Which of the following statements about vesicle budding from the Golgi is false?
(a)
Clathrin molecules are important for binding to and selecting cargos for
transport.
(b)
Adaptins interact with clathrin.
(c)
Once vesicle budding occurs, clathrin molecules are released from the
vesicle.
(d)
Clathrin molecules act at the cytosolic surface of the Golgi membrane.
15-26 Your friend has just joined a lab that studies vesicle budding from the Golgi and
has been given a cell line that does not form mature vesicles. He wants to start
designing some experiments but wasn’t listening carefully when he was told
about the molecular defect of this cell line. He’s too embarrassed to ask and
comes to you for help. He does recall that this cell line forms coated pits but
vesicle budding and the removal of coat proteins don’t happen. Which of the
following proteins might be lacking in this cell line?
(a)
clathrin
(b)
Rab
(c)
dynamin
(d)
adaptin
15-27 Which of the following protein families are not involved in directing transport
vesicles to the target membrane?
(a)
SNAREs
(b)
Rabs
(c)
tethering proteins
(d)
adaptins
15-28 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.
15-29 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. In
some cases, v-SNAREs and t-SNAREs may also mediate the 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 we have 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
consults with you regarding the interpretation of his data.
Your friend has designed an ingenious assay for the fusion of vacuolar vesicles by
using alkaline phosphatase. The protein alkaline phosphatase is made in a “pro”
form that must be cleaved 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), whereas 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 phosphatase, whose activity
can be measured and quantified.
Figure Q15-29
Your friend has taken each of these yeast strains and further engineered them so
that they express only the v-SNAREs, only the t-SNAREs, both SNAREs (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, by using the alkaline phosphatase assay. The data are
shown in the graph in Figure Q15-29B. On this graph, the SNARE present on the
vesicle of the particular yeast strain is indicated as “v” (for the presence of the vSNARE) and “t” (for the presence of the t-SNARE).
What do his data say about the requirements for v-SNAREs and t-SNAREs in the
vacuolar vesicles? Is it important to have a specific type of SNARE (that is, vSNARE or t-SNARE) on each vesicle?
Secretory Pathway
15-30 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 N-terminus of the protein.
(d)
the asparagine in the sequence Asn-X-Ser/Thr.
15-31 Name two types of protein modification that can occur in the ER but not in the
cytosol.
15-32 Which of the following statements about disulfide bond formation is false?
(a)
Disulfide bonds do not form under reducing environments.
(b)
Disulfide bonding occurs by the oxidation of pairs of cysteine side chains
on the protein.
(c)
Disulfide bonding stabilizes the structure of proteins.
(d)
Disulfide bonds form spontaneously within the ER because the lumen of
the ER is oxidizing.
15-33 Cells have oligosaccharides displayed on their cell surface that are important for
cell–cell recognition. Your friend discovered a transmembrane glycoprotein, GP1,
on a pathogenic yeast cell that is recognized by human immune cells. He decides
to purify large amounts of GP1 by expressing it in bacteria. To his purified
protein he then adds a branched 14-sugar oligosaccharide to the asparagine of the
only Asn-X-Ser sequence found on GP1 (Figure Q15-33). Unfortunately, immune
cells do not seem to recognize this synthesized glycoprotein. Which of the
following statements is a likely explanation for this problem?
Figure Q15.33
(a)
(b)
(c)
(d)
The oligosaccharide should have been added to the serine instead of the
asparagine.
The oligosaccharide should have been added one sugar at a time.
The oligosaccharide needs to be further modified before it is mature.
The oligosaccharide needs a disulfide bond.
15-34 If you remove the ER-retention signal from a protein that normally resides in the
ER lumen, where do you predict the protein will ultimately end up? Explain your
reasoning.
15-35 Which of the following statements about the unfolded protein response (UPR) is
false?
(a)
Activation of the UPR results in the production of more ER membrane.
(b)
Activation of the UPR results in the production of more chaperone
proteins.
(c)
Activation of the UPR occurs when receptors in the cytoplasm sense
misfolded proteins.
(d)
Activation of the UPR results in the cytoplasmic activation of gene
regulatory proteins.
15-36 Match the set of labels below with the numbered label lines on Figure Q15-36.
Figure Q15-36
A.
B.
C.
D.
E.
cisterna
Golgi stack
secretory vesicle
trans Golgi network
cis Golgi network
15-37 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. Table Q15-37
summarizes the results.
Table Q15-37
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.
2.
3.
4.
central Golgi cisternae
cis Golgi network
ER
trans Golgi network
15-38 Which of the following statements about secretion is true?
(a)
The membrane of a secretory vesicle will fuse with the plasma membrane
when it discharges its contents to the cell’s exterior.
(b)
Vesicles for regulated exocytosis will not bud off the trans Golgi network
until the appropriate signal has been received from the cell.
(c)
The signal sequences of proteins destined for constitutive exocytosis
ensure their packaging into the correct vesicles.
(d)
Proteins destined for constitutive exocytosis aggregate as a result of the
acidic pH of the trans Golgi network.
15-39 For each of the following sentences, choose one of the two 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.
15-40 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?
Endocytic Pathways
15-41 You are working in a biotech company that has discovered a small-molecule drug
called H5434. H5434 binds to LDL receptors when they are bound to cholesterol.
H5434 binding does not alter the conformation of the LDL receptor’s intracellular
domain. Interestingly, in vitro experiments demonstrate that addition of H5434
increases the affinity of LDL for cholesterol and prevents cholesterol from
dissociating from the LDL receptor even in acidic conditions. Which of the
following is a reasonable prediction of what may happen when you add H5434 to
cells?
(a)
Cytosolic cholesterol levels will remain unchanged relative to normal
cells.
(b)
Cytosolic cholesterol levels will decrease relative to normal cells.
(c)
The LDL receptor will remain on the plasma membrane.
(d)
The uncoating of vesicles will not occur.
15-42 Name three possible fates for an endocytosed molecule that has reached the
endosome.
15-43 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 uses
__________________ proteins to form small vesicles containing
fluids and molecules. After these vesicles have pinched off from
the plasma membrane, they will fuse with the
__________________, where materials that are taken into the
vesicle 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
cholesterol
Golgi apparatus
mycobacterium
pseudopods
rough ER
clathrin
endosome
phagocytosis
pinocytosis
SNARE
transcytosis
15-44 Fibroblast cells from patients W, X, Y, and Z, each of whom has 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.
2.
3.
a defect in one of the lysosomal hydrolases
a defect in the phosphotransferase that is required for mannose-6phosphate tagging of the lysosomal hydrolases
a defect in the mannose-6-phosphate receptor, which binds mannose-6phosphate-tagged lysosomal proteins in the trans Golgi network and
delivers them to lysosomes
When you incubate some of these mutant fibroblasts in a medium in which
normal cells have been grown, you find that the inclusion bodies disappear.
Because of these results, you suspect that the constitutive exocytic pathway in
normal cells is secreting lysosomal hydrolases that 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 medium from
normal cells and medium from each of the other mutant cell cultures, and get the
results summarized in Table Q15-44.
Table Q15-44
Indicate which defect (1, 2, 3) each patient (W, X, Y, Z) is most likely to have.
15-45 If a lysosome breaks, what protects the rest of the cell from lysosomal enzymes?
How We Know: Tracking Protein and Vesicle Transport
15-46 You have created a GFP fusion to a protein that is normally secreted from yeast
cells. Because you have learned about the use of temperature-sensitive mutations
in yeast to study protein and vesicle transport, you obtain three mutant yeast
strains, each 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 to 4, and then you carry
out the experiment anyway, obtaining the results shown in Figure Q15-46 (the
black dots represent your GFP fusion protein).
Figure Q15-46
Name the process that is defective in each of these strains. Remember that one of
these strains is your wild-type control.