Case 6

● When a protein folds into a compact

structure, it buries most of its hydrophobic
residues in an interior core. In addition,
large numbers of noncovalent interactions
form between various parts of the
molecule. It is the sum of all of these
energetically favorable arrangements that
determines the final folding pattern of the
polypeptide chain—as the conformation of
lowest free energy.
● Experiments have demonstrated that once
a protein domain in a multi-domain protein
emerges from the ribosome, it forms a
compact structure within a few seconds
that contains most of the final secondary
structure (α helices and β sheets) aligned
in roughly the right way
● For many protein domains, this unusually
open and flexible structure, which is called
a molten globule, is the starting point for a
relatively slow process in which many side-
chain adjustments occur that eventually
form the correct tertiary structure.
Nevertheless, because it takes several
minutes to synthesize a protein of average
size, a great deal of the folding process is
complete by the time the ribosome
releases the C-terminal end of a protein
● Molecular chaperones were first identified
in bacteria when E. coli mutants that failed
to allow bacteriophage lambda to replicate
in them were studied. These mutant cells
produce slightly altered versions of the
chaperone machinery, and as a result they
are defective in specific steps in the
assembly of the viral proteins.

● The molecular chaperones are included

among the heat-shock proteins (hence
their designation as hsp), because they are
synthesized in dramatically increased
amounts after a brief exposure of cells to
an elevated temperature (for example,
42°C for cells that normally live at 37°C).
This reflects the operation of a feedback
system that responds to any increase in
misfolded proteins (such as those produced
by elevated temperatures) by boosting the
synthesis of the chaperones that help these
proteins refold. ERAD

● Eucaryotic cells have at least two major

families of molecular chaperones—known
as the hsp60 and hsp70 proteins. Different
family members function in different
organelles. Mitochondria contain their own
hsp60 and hsp70 molecules that are
distinct from those that function in the
 cytosol, and a special hsp70 (called BIP)
helps to fold proteins in the endoplasmic
reticulum.

● The hsp60-like and hsp70 proteins each

work with their own small set of associated
proteins when they help other proteins to
fold. They share an affinity for the exposed
hydrophobic patches on incompletely
folded proteins, and they hydrolyze ATP,
often binding and releasing their protein
with each cycle of ATP hydrolysis.

● The hsp70 machinery acts early in the life

of many proteins, binding to a string of
about seven hydrophobic amino acids
before the protein leaves the ribosome
(Figure 6-83). In contrast, hsp60-like
proteins form a large barrel-shaped
structure that acts later in a protein's life,
after it has been fully synthesized. This
type of chaperone forms an “isolation
chamber” into which misfolded proteins are
fed, preventing their aggregation and
providing them with a favorable
environment in which to attempt to refold
●

● Many of the cell's polysaccharides are

made in the Golgi apparatus, including the
pectin and hemicellulose of the cell wall in
plants and most of the glycosaminoglycans
of the extracellular matrix in animals

● But the Golgi apparatus also lies on the

exit route from the ER, and a large
proportion of the carbohydrates that it
makes are attached as oligosaccharide side
chains to the many proteins and lipids that
the ER sends to it.

● A subset of these oligosaccharide groups

serve as tags to direct specific proteins into
vesicles that then transport them to
 lysosomes. But most proteins and lipids,
once they have acquired their appropriate
oligosaccharides in the Golgi apparatus,
are recognized in other ways for targeting
into the transport vesicles going to other
destinations.

● Proteins that have entered the ER and

are destined for the Golgi apparatus or
beyond are first packaged into small
COPII-coated transport vesicles. These
 transport vesicles bud from specialized
regions of the ER called ER exit
sites, whose membrane lacks bound
ribosomes. In most animal cells, ER exit
sites seem to be randomly dispersed
throughout the ER network.
● The exit signals that direct proteins out of
the ER for transport to the Golgi and
beyond are mostly not understood. There
is one exception, however. The ERGIC53
protein seems to serve as a receptor for
packaging some secretory proteins into
COPII-coated vesicles. Its role in protein
transport was identified because humans
who lack it owing to an inherited
mutation have lowered serum levels of
two secreted blood-clotting factors
(Factor V and Factor VIII) and therefore
bleed excessively. The ERGIC53 protein is
a lectin that binds mannose and is
thought to recognize this sugar on Factor
V and Factor VIII proteins, thereby
packaging the proteins into transport
vesicles in the ER.
●

In step 1, identical pairs of v-SNAREs and

t-SNAREs in both membranes are pried
apart by NSF (see Figure 13-13). In steps
2 and 3, the separated matching SNAREs
on adjacent identical membranes
interact, which leads to membrane fusion
and the formation of one continuous
compartment. Subsequently, the
compartment grows further by homotypic
fusion with vesicles from the same kind
of membrane, displaying matching
SNAREs. Homotypic fusion is not
restricted to the formation of vesicular
tubular clusters; in a similar process,
endosomes fuse to generate larger
endosomes. Rab proteins help regulate
the extent of homotypic fusion and hence
 the size of the compartments in a cell
(not shown).
● The clusters are relatively short-lived
because they quickly move along
microtubules to the Golgi apparatus,
where they fuse and deliver their
contents
● As soon as vesicular tubular clusters
form, they begin budding off vesicles of
their own. Unlike the COPII-coated
vesicles that bud from the ER, these
vesicles are COPI-coated. They carry
back to the ER resident proteins that
have escaped, as well as proteins that
participated in the ER budding reaction
and are being returned.
● This retrieval process demonstrates the
exquisite control mechanisms that
regulate coat assembly reactions. The
COPI coat assembly begins only seconds
after the COPII coats have been shed. It
remains a mystery how this switchover in
coat assembly is controlled.
● A similar retrieval process continues
from the Golgi apparatus, after the
vesicular tubular clusters have delivered
their cargo.

Golgi Apparatus
● It consists of a collection of flattened,
membrane-enclosed cisternae, somewhat
resembling a stack of pancakes. Each of
these Golgi stacks usually consists of four
to six cisternae (Figure 13-22), although
some unicellular flagellates can have up
to 60. In animal cells, many stacks are
linked by tubular connections between
corresponding cisternae, thus forming a
single complex, which is usually located
near the cell nucleus and close to the
centrosome (Figure 13-23A).
● This localization depends on
microtubules. If microtubules are
experimentally depolymerized, the Golgi
apparatus reorganizes into individual
stacks that are found throughout the
cytoplasm, adjacent to ER exit sites. In
some cells, including most plant cells,
hundreds of individual Golgi stacks are
normally dispersed throughout the
cytoplasm.
●
●

● Proteins exported from the ER enter the

first of the Golgi processing
compartments (the cis Golgi
compartment), after having passed
through the cis Golgi network. They then
move to the next compartment
(the medial compartment, consisting of
the central cisternae of the stack) and
finally to the trans compartment, where
glycosylation is completed. The lumen of
the trans compartment is thought to be
continuous with the trans Golgi network,
where proteins are segregated into
different transport packages and
dispatched to their final destinations—the
 plasma membrane, lysosomes, or
secretory vesicles.
● the stack forms a multistage processing
unit.
● This compartmentalization might seem
unnecessary, since each oligosaccharide
processing enzyme can accept a
glycoprotein as a substrate only after it
has been properly processed by the
preceding enzyme.
●

The localization of each processing step shown

was determined by a combination of techniques,
including biochemical subfractionation of the
Golgi apparatus membranes and electron
microscopy after staining with antibodies specific
for some of the processing enzymes. The
locations of many other processing reactions
have not been determined. Although only three
distinguishable cisternal compartments have so
far been demonstrated, each of these sometimes
consists of a group of two or more cisternae in
sequence. It is likely that each processing
enzyme is not completely restricted to a
particular cisterna but that its distribution is
graded across the stack—such that early acting
enzymes are present mostly in the cis Golgi
cisternae and later acting enzymes are mostly in
the trans Golgi cisternae.

It is likely that the transport through the Golgi

apparatus in the forward direction (red
arrows) involves elements of both of the views
represented here. (A) In the vesicular transport
model, Golgi cisternae are static organelles,
which contain a characteristic complement of
resident enzymes. The passing of molecules
through the Golgi is accomplished by forward-
moving transport vesicles, which bud from one
cisterna and fuse with the next in a cis-to-
trans direction. (B) According to the alternative
cisternal maturation model, each Golgi cisterna
matures as it migrates outwards through a stack.
At each stage, the Golgi resident proteins that
are carried forward in a cisterna are moved
backward to an earlier compartment in COPI-
coated vesicles. When a newly formed cisterna
moves around to a medial position, for example,
“left-over” cis Golgi enzymes would be extracted
and transported backward to a new cis cisterna
behind. Likewise, the medial enzymes would be
received by retrograde transport from the
cisternae just ahead. In this way, a cis cisterna
would mature to a medial cisterna as it moves.

The vesicular transport and the cisternal

maturation model are not mutually exclusive.
Indeed, evidence suggests that transport may
occur by a combination of the two mechanisms,
in which some cargo is moved forward rapidly in
transport vesicles, whereas other cargo is moved
forward more slowly as the Golgi apparatus
constantly renews itself through cisternal
maturation.

Gaucher disease, the most prevalent

lysosomal storage disorder, presents with
an elevated incidence among Ashkenazi
Jews. It is an autosomal recessive inborn
error of metabolism characterized by the
toxic accumulation of glucocerebroside
lipids within multiple organs. Gaucher
disease results from mutations in
the GBA1 gene, leading to deficient
glucocerebrosidase activity within
lysosomes. Clinical manifestations,
including hepatosplenomegaly,
pancytopenia, osteoporosis, and avascular
necrosis, vary in severity depending on the
disease type. This activity comprehensively
examines the assessment and management
of Gaucher disease while emphasizing the
pivotal role of the interprofessional team in
collaborating and delivering well-
coordinated care to improve patient
outcomes.

I-cell disease (ML-II) and pseudo-Hurler

polydystrophy (ML-III), biochemically
related diseases, are caused by failure in
the transport of soluble
lysosomal enzymes from the Golgi
apparatus into the lysosome. Targeting of
lysosomal enzymes from the Golgi
apparatus to lysosomes is mediated by
receptors that bind mannose-6-phosphate
recognition markers on the enzymes. N-
Acetylglucosamine-1-phosphotransferase,
the enzyme that catalyzes the
phosphorylation of mannose, is composed
of three subunits α, β, and γ coded by two
different genes. Mutations in the α/β-
subunit precursor gene result in ML-II and
in classical ML-III (ML-IIIA), and mutations
in subunit γ result in ML-III.

Case 7

● Most mitochondrial proteins are translated on

free cytosolic ribosomes and imported into
the organelle by specific targeting signals. In
addition, mitochondria are unique among the
cytoplasmic organelles already discussed in
that they contain their own DNA, which
encodes tRNAs, rRNAs, and some
mitochondrial proteins.

● The matrix contains the mitochondrial

genetic system as well as the enzymes
 responsible for the central reactions of
oxidative metabolism

●
● First, its surface area is substantially
increased by its folding into cristae. In
addition, the inner mitochondrial membrane
contains an unusually high percentage
(greater than 70%) of proteins, which are
involved in oxidative phosphorylation as well
as in the transport of metabolites (e.g.,
pyruvate and fatty acids) between the
cytosol and mitochondria.
● Otherwise, the inner membrane is
impermeable to most ions and small
molecules—a property critical to maintaining
the proton gradient that drives oxidative
phosphorylation.
● Outer membrane -> Porins -> 6000 daltons
● This hypothesis has recently been
substantiated by the results of DNA sequence
analysis, which revealed striking similarities
between the genomes of mitochondria and of
the bacterium Rickettsia prowazekii.
● Rickettsia are intracellular parasites which,
like mitochondria, are only able to reproduce
within eukaryotic cells. Consistent with their
similar symbiotic lifestyles, the genomic DNA
sequences of Rickettsia and mitochondria
suggest that they share a common ancestor,
from which the genetic system of present-
day mitochondria evolved.
● Mitochondrial genomes are usually circular
DNA molecules, like those of bacteria, which
are present in multiple copies per organelle.
They vary considerably in size between
different species. The genomes of human and
most other animal mitochondria are only
about 16 kb, but substantially larger
mitochondrial genomes are found in yeasts
(approximately 80 kb) and plants (more than
200 kb).
● For example, the largest sequenced
mitochondrial genome is that of the
plant Arabidopsis thaliana.
Although Arabidopsis mitochondrial DNA is
approximately 367 kb, it encodes only 32
proteins: just more than twice the number
encoded by human mitochondrial DNA. The
largest number of mitochondrial genes has
been found in mitochondrial DNA of the
protozoan Reclinomonas americana, which is
69 kb and contains 97 genes.
●
● The genome contains 13 proteincoding
sequences, which are designated as
components of respiratory complexes I, III, IV,
or V. In addition, the genome contains genes
for 12S and 16S rRNAs and for 22 tRNAs,
which are designated by the one-letter code
for the corresponding amino acid. The region
of the genome designated “D loop” contains
an origin of DNA replication and
transcriptional promoter sequences.

Protein Import and Mitochondrial

Assembly
In contrast to the RNA components of the
mitochondrial translation apparatus (rRNAs
and tRNAs), most mitochondrial genomes
do not encode the proteins required for
DNA replication, transcription, or
translation. Instead, the genes that
encode proteins required for the
replication and expression of
mitochondrial DNA are contained in the
nucleus. In addition, the nucleus
contains the genes that encode most of
the mitochondrial proteins required for
oxidative phosphorylation and all of the
enzymes involved in mitochondrial
metabolism (e.g., enzymes of the citric acid
cycle). The proteins encoded by these
genes (more than 95% of mitochondrial
proteins) are synthesized on free
cytosolic ribosomes and imported into
mitochondria as completed polypeptide
chains.
●
● 20 to 35 amino acids (called presequences)
● The polypeptide chains are then inserted
into a protein complex that directs
translocation across the outer membrane
(the translocase of the outer membrane or
Tom complex).
● The proteins are then transferred to a second
protein complex in the inner membrane (the
translocase of the inner membrane or Tim
complex). Continuing protein translocation
requires the electrochemical potential
established across the inner mitochondrial
membrane during electron transport.

● As discussed in the next section of this

chapter, the transfer of high-energy electrons
from NADH and FADH2 to molecular oxygen is
coupled to the transfer of protons from the
mitochondrial matrix to the intermembrane
space. Since protons are charged particles,
this transfer establishes an electric potential
across the inner membrane, with the matrix
being negative. During protein import, this
electric potential drives translocation of the
positively charged presequence.

● These proteins are targeted to their

destinations by a second sorting signal
following the positively charged presequence
that directs mitochondrial import. The
targeting of proteins to the mitochondrial
membranes appears to be mediated by
hydrophobic stop-transfer sequences that
halt translocation of the polypeptide chains
through the Tim or Tom complexes, leading
to their insertion into the inner or outer
mitochondrial membranes, respectively

DNP

In humans, DNP causes dose-dependent

mitochondrial uncoupling, causing the rapid
loss of ATP as heat and leading to
uncontrolled hyperthermia—up to 44 °C (111
°F)—and death in case of overdose.