Download 5-Cell and Molecular Biology (Golgi etc)

 Golgi Apparatus:
 Introduction;
 The Golgi apparatus also called Golgi complex is usually located near
the cell nucleus and
 In animal cells it is often close to the centrosome or cell center
 It consists of a collection of flattened, membrane-bound cisternae and
 Thus resembles a stake of a plates
 Each of these Golgi stakes usually consists of four to six cisternae
Fig 13.4 Alberts 3rd Ed
 The number of Golgi stakes per cell varies greatly depending on the cell
type;
• Some animal cells contain one large stake, while certain plant cells
contain hundreds of small ones
• Groups of small vesicles are associated with the Golgi stakes,
clustered on the side next to the ER and along the dilated rims of each
cisterna
 These Golgi vesicles are thought to transport proteins and lipids both to
and from the Golgi apparatus and between the Golgi cisternae
 During their passage through the Golgi apparatus, the transported
molecules undergo an ordered series of covalent modifications
 Each Golgi stack has two distinct faces:
• a cis face (entry face) and
• a trans face (or exit face)
 Both cis and trans faces are closely connected to special compartments
 Which are composed of a network of interconnected tubular and cisternal
structures
 These are the cis Golgi network also called the intermediary or salvage
compartment and
 The trans Golgi network, respectively
 Proteins and lipids
• enter the cis Golgi network in transport vesicles from the ER and
• exit from the trans Golgi network in transport vesicles destined for the
cell surface or another compartment
 Both networks are thought to be important for protein sorting:
• Proteins entering the cis Golgi network can either move onward in the
Golgi apparatus or be returned to the ER
• Proteins existing the trans Golgi network are sorted according to
whether they are destined for lysosomes, secretory vesicles or the cell
face
• The Golgi apparatus is specially prominent in cells that are
specialized for secretion. Such as
 The globlet cells of the intestinal epithelium which secrete large
amounts of polysaccharide-rich mucus in to the gut
 In such cells, unusually large vesicles are found on the trans side
of Golgi apparatus
 Which faces the plasma membrane domain where secretion occurs
Fig 13.5 Alberts 3rd Ed
 ER-Resident Proteins are Selectively Retrieved from the Cis Golgi
Network;
 Vesicles destined for the Golgi apparatus bud from a specialized region
of the ER called the transitional elements
• Whose membrane lacks bound ribosomes and
• is often located between the rough ER and the Golgi apparatus
Fig 13.6 Alberts 3rd Ed
 Vesicles budding from the transitional elements of the ER are thought to
be non-selective
 They will transport any protein in the ER to the Golgi apparatus,
although it remains possible that there are signals which accelerate the
process
 However, there is one strict requirement for the exit of a protein from the
ER
• It must be correctly folded and assembled
• Proteins that misfolded or incompletely assembled in to their protein
complexes are retained in the ER, either
 Bound to the special binding protein BiP or
 In aggregates that can not be packaged and
 are eventually degraded with in ER
• Thus exit from the ER can be regarded as a quality check point means:
 Protein would be discarded unless folding and subunit assembly are
successfully completed
• In fact, the ER seems to be one of the main sites in the cell where
proteins are degraded while other is lysosomes
 Therefore, correctly folded proteins do not need a special signal to be
transported out of the ER
 But those that are resident in the ER lumen such as BiP do need such a
signal to be retained there
 Retention of soluble ER-resident proteins is mediated by a short, four
amino acid sorting signal, identified as KDEL (Lys-Asp-Glu-Lue) or a
similar sequence
Table 12.3 Alberts 3rd Ed
 By genetic engineering, if this retention signal has been removed from
BiP, then BiP is secreted from the cell
 Similarly, if signal is transferred to a protein that is normally secreted, the
protein is now retained in the lumen of ER
 The retention signal works not by anchoring resident proteins in the lumen
of the ER
 But by selective retrieval of ER-resident proteins after they have escaped
in transport vesicles and been delivered to the cis Golgi network
 In the cis Golgi network, a specific membrane-bound receptor protein
binds to the ER retention signal and
 Packages any proteins displaying the signal into special transport vesicles
that return the proteins to the ER
 Thus for these resident proteins the ER is like an open prison: there is
nothing to stop them leaving, but if they leave, they are brought back
Fig 13.7 Alberts 3rd Ed
 Golgi Proteins Return to the ER When Cells are Treated with the Drug;
 The continuous retrieval of ER-resident proteins from cis-Golgi network
means that transport between these two organelles occurs in both
directions
 As mentioned in Fig 13.7, receptors for the ER retention signal are also
found in the later Golgi compartments, suggesting that a return pathway
from these compartments to the ER exists
 The importance of return pathway from Golgi to ER is dramatically
illustrated by studies using the drug brefeldin A
• Which blocks protein secretion by disrupting the Golgi apparatus
 In brefeldin A treated cells, the Golgi apparatus largely disappears and
the Golgi proteins end up in the ER
•
where they intermix with ER proteins
 When the drug is removed, the normal Golgi apparatus reforms and the
Golgi proteins return to their proper Golgi compartments
Fig 13.8 Alberts 3rd Ed
Fig 13.9 Alberts 3rd Ed
 Oligosaccharide Chains are Processed in the Golgi Apparatus;
 As discussed earlier, a single species of N-linked oligosaccharide is
attached en block to many protein in the ER
 This oligosaccharide is then trimmed while protein is still in the ER
Fig 12.48 Alberts 3rd Ed
Fig 12.49 Alberts 3rd Ed
 Further modifications and additions occur in the Golgi apparatus,
depending on the protein
 The outcome is that two broad classes of N-linked oligosaccharides are
found attached to mammalian glycoproteins:
• The complex oligosaccharides and
• The high mannose oligosaccharides
 The complex oligosaccharides can contain:
• more than the original two N-acetylglucosamines as well as
• a variable number of galactose and
• sialic acid residues and in some cases, fucose
• Sialic acid is of special importance because it is the only sugar in
glycoproteins that contains a net negative charge
Fig 13.10 Alberts 3rd Ed
 High-mannose oligosaccharides have
• no new sugars added to them in the Golgi apparatus
•
they contain just two N-acetylglucosamine and many mannose
residues
 The complex oligosaccharides are generated by a combination of
trimming the original oligosaccharide added in the ER and
 The addition of further sugars
 The processing that generates complex oligosaccharide chains follows
the highly ordered pathway
Fig 13.11 Alberts 3rd Ed
Fig 13.12 Alberts 3rd Ed
 The Golgi Cisternae are Organized as a Series of Processing
Compartments;
 Proteins exported from the ER enter the first of the Golgi processing
compartments - the cis compartment - which is thought to be continuous
with the cis Golgi network
 Then they moved to next compartment - the medial compartment,
consisting of the central cisternae of 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 in to different transport vesicles and
dispatched to their final destinations i.e.
• Plasma membrane
• Lysosomes or
• Secretory vesicles
 These oligosaccharide processing pathways occur in a correspondingly
organized sequence in the Golgi stack, with each cisterna containing its
own set of processing enzymes
 Proteins are modified in successive stages as they move from cisterna to
cisterna across the stack
 So that the stack forms a multistage processing unit
 The processing occurs in a spatial as well as a biochemical sequence:
• Enzymes catalyzing early processing steps are localized in cisternae
toward the cis face of Golgi stake
• Whereas enzymes catalyzing later processing steps are localized in
cisternae toward the trans face
 The transport of proteins between different Golgi cisternae is thought to
be mediated by transport vesicles
• Which bud from one cisterna and fuse with the next
Fig 13.13 Alberts 3rd Ed
Fig 13.14 Alberts 3rd Ed
 Proteoglycanes are Assembled in the Golgi Apparatus;
 It is not only the N-linked oligosaccharide chains on proteins that are
altered as the protein pass through the Golgi cisternae en route from the
ER to their final destinations

Many other proteins are also modified in other ways. For example:
• Some have sugars added to the OH groups of selected serine or
threorine side chains
• This O-linked glycosylation is catalyzed by a series of glycosyl
transferase enzymes
• That use the sugar nucleotides in the lumen of the Golgi apparatus to
add sugar residues to a protein one at a time
• Usually, N-acetylgalatosamine is added first followed by a variable
number of additional sugar residues
 ranging from just a few to 10 or more
 The Golgi apparatus confer the heaviest glycosylation of all on
proteoglycan core proteins
• which it modifies to produce proteoglycan
• this process involves the polymerization of one or more
glycosaminoglycan chains via a xylose link on to serines on the core
protein
• Many proteoglycanes are secreted and become components of the
extracellular matrix while other remain anchored to the plasma
membrane
• Others form a major component of slimy materials such as the mucus
that is secreted to form a protective coating over many epithelia
• The sugars incorporated in to glycosaminoglycanes are heavily
sulfated in the Golgi apparatus immediately after these polymers are
made and
• This helps to give proteoglycans their negative charge
Fig 19.36 Alberts 3rd Ed
 The Carbohydrates in Cell Membrane Faces the Side of the Membrane
That is Topologically Equivalent to the Outside of the Cell;
 Because all oligosaccharide chains are added on the luminal side of the
ER and Golgi apparatus, the distribution of carbohydrate on membrane
proteins and lipids is asymmetrical
(Glycosaminoglycan –
unbranched polysaccharide
chains composed of repeating
disaccharide units either Nacetylglucosamine or Nacetylgalactosamine – an
amino sugar and second one
is usually uronic acid)
 Similar to asymmetry of the lipid bilayer, the asymmetric orientation of
these glycosylated molecules is maintained during their transport to the:
• plasma membrane
• secretory vesicles or
• lysosomes
 As a result, the oligosaccharides of all of the glycoproteins and
glycolipids in the corresponding intracellular membranes face the lumen
 While those in the plasma membrane face the outside of the cell
Fig 13.15 Alberts 3rd Ed
 What is the Purpose of Glycosylation?
 There is an important difference between the construction of
oligosaccharide and the synthesis of other macromolecules such as
• DNA
• RNA and
• Proteins
 DNA and RNA and proteins are copied from a template in a repeated
series of identical steps using the same enzyme(s)
 While complex carbohydrates require a different enzyme at each step
• Each product being recognized as the exclusive substrate for the next
enzyme in the series
 Since the complicated pathways have evolved to synthesize them, it
seems likely that
• The oligosaccharides on glycolipids and glycoproteins have
important functions which are mostly unknown
 N-linked glycosylation is prevalent in all eukaryotes including yeast but
absent from prokaryotes
 N-linked oligosaccharides also occur in a very similar form in archaeal
cell wall proteins
 Suggesting that the whole machinery required for their synthesis is
evolutionarily ancient
 N-linked glycosylation promotes protein folding in two ways:
i.
It had direct role in making folding intermediates more soluble,
thereby preventing their aggregation
ii.
The sequential modifications of the N-linked oligosaccharides
establish a “glyco code” that marks
o
the progression of protein folding and
o mediates the binding of the protein to chaperones and lectins
(carbohydrate binding proteins). For example:
-
In guiding ER to Golgi transport
-
Lectins also participate in protein sorting in the trans
Golgi network
 Besides, because chains of sugar have limited flexibility, they can limit
the approach of other macromolecules to the protein surface
Fig 13.16 Alberts 3rd Ed
• In this way, a glycoprotein more resistant to digestion by proteolytic
enzymes
• It may be that oligosaccharides on cell surface proteins originally
provided an ancestral cell with protective coat
• Compared to the rigid bacterial cell wall, a mucus coat has the
advantage that it leaves the cell with the freedom to change shape and
move. For example:
 Mucus coat of lung and intestinal cells protects against many
pathogens
 The recognition of sugar chains by lectins in the extracellular
space is important in many developmental processes and in cellcell recognition. For example:
o Selectins (cell adhesion molecules) are lectins that function in
cell-cell adhesion during lymphocyte migration
• Presence of oligosaccharides may modify a protein’s antigenic
properties, making glycosylation an important factor in the
production of proteins (antibodies) for pharmaceutical purposes
• Glycosylation can also have important regulatory roles. For example:
 Signaling through the cell-surface signaling receptor - Notch
determines the cell’s fate in development
Fig P 600 Alberts 3rd Ed
 Lysosomes:
 Introduction;
 Lysosomes are membranous bags of hydrolytic enzymes used for the
controlled intracellular digestion of macromolecules
 They contain about 40 types of hydrolytic enzymes including:
• Proteases
• Nucleases
• Glycosidases
• Lipases
• Phospholipases
• Phophatases and
• Sulfatases
 All are hydrolyses and for optimal activity they require an acid
environment and
 Lysosome provides this by maintaining pH of about 5 in its interior
 In this way, the content of cytosol are doubly protected against attack by
the cell’s own digestive system
 The membrane of lysosome normally keeps the digestive enzymes out of
the cytosol
 But even if they leak out, they can do little damage at the cytosolic pH of
about 7.2
 Like all other intracellular organelles, the lysosome not only contains a
unique collection of enzymes
 But also has a unique surrounding membrane
 Transport proteins in this membrane allow the final products of the
digestion of macromolecules such as
• amino acids
• sugars and
• nucleotides to be transported to the cytosol from where they can be
excreted or reutilized by the cell
• An H+ pump in the lysosomal membrane utilizes the energy of ATP
hydrolysis to pump H+ into lysosome
• Thereby maintaining the lumen at its acidic pH
Fig 13.17Alberts 3rd Ed
• Most of the lysosomal membrane proteins are unusually highly
glycosylated
• Which is thought to help in protecting them from the lysosomal
proteases in the lumen
• As discussed earlier, endocytosed materials are initially delivered to
organelles called endosomes before being delivered to lysosomes
• Endosomes also have H+ pumps that keep their lumen at a low pH
though not as low as that of lysosomes
Fig 13.18Alberts 3rd Ed
• Lysosomes were initially discovered by biochemical fractionations of
cell extracts
• Later on they were seen clearly in the electron microscope
• They are extraordinarily diverse in shape and size
• but can be identified as members of a single family of organelles by
histochemistry
 using the precipitate produces by the action of an acid hydrolase on its
substrate to show
 which organelles contain the enzyme
Fig 13.19 Alberts 3rd Ed
• By this criterion, lysosomes are found in all eukaryotic cells
• The heterogeneity of lysosomal morphology contrasts with the relatively
uniform structures of most other cellular organelles
• The diversity reflects the wide variety of digestive functions mediated by
acid hydrolyses including:
 The break down of intra and extracellular debris
 The destruction of phagocytosed microorganisms and
 The production of nutrients for the cell
 Plant and Fungal Vacuoles are Remarkably Versatile
Lysosomes;
 Most plant and fungal cells including yeasts contain one or several very
large, fluid-filled vesicles called vacuoles
 They typically occupy more than 30% of the cell volume and as much
as 90% in some cell types
Fig 13.20 Alberts 3rd Ed
 Vacuoles are related to lysosomes of animal cells containing a variety of
hydrolytic enzymes
 But their functions are remarkably diverse
 The plant vacuole can act:
• as a storage organelle for nutrients and for waste products
• as a degradative compartment
• as an economical way of increasing cell size and as a controller of
turgor pressure – the osmotic pressure that pushes outward on the cell
wall and keeps the plant from wilting (loss of rigidity of non-woody
parts of plants)
Fig 13.21 Alberts 3rd Ed
• Different vacuoles with distinct functions i.e. digestion and storage
are often present in the same cell
 Vacuole is important as a homeostatic device enabling plant cells to
withstand wide variations in their environment
 When the pH in the environment drops. For example:
• The flex of H+ in to the cytosol is balanced, at least in part, by
increased transport of H+ in to the vacuole so as to keep the pH in the
cytosol constant
• Similarly, many plant cells maintain an almost constant turgor
pressure in the face of large changes in the tonicity (it is a measure of
the osmotic pressure gradient of two solutions separated by a semi
permeable membrane) of the fluid in their immediate environment
• They do so by changing the osmotic pressure of the cytosol and
vacuole - in part by controlled breakdown and re-synthesis of
polymers such as
 polyphosphate in the vacuole and in part by
 altering rates of transport of sugars, amino acids and other
metabolites across the plasma membrane and
 vacuolar membrane
• Substances stored in the plant vacuoles in different species range from
rubber to opium to the flavoring of garlic
• Often stored products have a metabolic function. For example;
 Proteins can be preserved for years in the vacuoles of the storage
cells of many seeds such as those of peas and beans
 When the seeds germinate, the proteins are hydrolyzed and the
mobilized amino acids provide a food supply for the developing
embryo
 Anthocyanin pigments that are stored in vacuoles, color the petals of
many flowers to attract pollination insects
 While noxious molecules that are released from vacuoles when a
plant is eaten or damaged provide a defense against predators
 Materials are Delivered to Lysosomes by Multiple Pathways;
 Lysosomes in general are meeting places in which several streams of
intracellular traffic converge
 A route that leads outward from the ER via the Golgi apparatus delivers
most digestive enzymes
 While at least three paths from different sources feed substances in to
lysosomes for digestion
i. The best studies is that followed by macromolecules taken up from
the external medium by endocytosis
ii. Degradation in lysosomes is used in all cell types for disposal of
obsolete parts of the cell itself – autophagy
iii. Provides materials to lysosomes for degradation occurs mainly in
cells specialized for the phagocytosis of large particle and
microorganisms
Fig 12.22 Alberts 3rd Ed
 Some Cytosolic Proteins are Directly Transported in to Lysosomes for
Degradation;
 There may be a fourth pathway for proteins to enter a lysosome for
degradation:
• Some proteins contain certain signal on their surface called KFERQ
sequences where
 K for lysine
 F for phenylalanine
 E for glutamate
 R for arginine and
 Q for glutamine
-
In liver cell, an average mitochondrion has a lifetime of
about 10 days
The process seems to begin with the enclosure of an
organelle by membrane derived from ER
 Because of this sequence, proteins will be delivered selectively to
lysosomes for degradation
 Lysosomal Enzymes are Sorted from Other Proteins in the Trans Golgi
Network by a Membrane-Bound Receptor Protein that Recognizes
Mannose -6-Phosphate;
 We now consider the system that delivers the other half of the traffic in to
lysosomes i.e.
• Specialized lysosomal hydrolases and
• Membrane proteins
 Both are synthesized in the rough ER and transported through the Golgi
apparatus
 Transport vesicles bud from the trans Golgi network incorporating
lysososmal proteins
 While excluding the many other proteins being packaged in to different
transport vesicles for delivery elsewhere
 The question is how lysosomal proteins are recognized and selected with
required accuracy?
Fig 13.14 Alberts 3rd Ed
 Mannose -6-phosphate (M6P) groups are recognized by their receptor
- For lysosomal hydrolases, the answer is
known
-They carry a unique marker in the form
of mannose-6-PO4
-That is added exclusively to the Nlinked oligosaccharides of these soluble
lysosomal enzymes
 Which are transmembrane proteins present in the trans Golgi network
 These proteins bind to the lysosomal enzymes and help package them in
to specific transport vesicles
 That then bud from the Golgi network and subsequently fuse with a late
endosome delivering their contents to the lumen of lysosomes
 The Mannose-6-Phospahte Receptor Shuttles Back and Forth Between
Specific Membranes;
 The M6P receptor binds its specific oligosaccharide at pH 7 in the trans
Golgi network and releases it at pH 6
 Which is the pH in the interior of late endosomes
 Thus the lysosomal hydrolases dissociate from the M6P in the late
endosomes and
 begin to digest the endocytosed material delivered from early endosomes
Fig 13.23 Alberts 3rd Ed
 A Signal Patch in the Polypeptide Chain Provides the Clue for Tagging a
Lysosomal Enzyme with Mannose 6-Phosphate;
 Lysosomal hydrolases not only contains N-linked oligosaccharides with
terminal mannose residues which is the site for addition of M6P group
but also a signal patch in its conformation
Fig 13.25 Alberts 3rd Ed
 Two enzymes act sequentially to catalyze the addition of M6P groups to
lysosomal hydrolases
Fig 13.24 Alberts 3rd Ed
 Defects in the GlcNAc Phosphotransferase Cause a Lysosomal Storage
Disease in Human;
 Lysosomal storage diseases are caused by genetic defects that affect one
or more of the lysosomal hydrolases and
 Result in accumulation of their undigested substrates in lysosomes with
sever pathological consequences. For example:
• Hurler’s disease - the enzyme
glycosaminoglycanes is missing
required
for
break
down
 So in these mutant individuals, the lysosomes accumulate massive
quantities of glycosaminoglycanes as these can not be digested
• Inclusion cell disease (I-cell disease) - almost all the hydrolytic
enzymes are missing from the lysosomes of fibroblasts and their
undigested substrates accumulate in lysosomes which consequently
form large “inclusions” in the patients
 In these individuals all the hydrolases missing from lysosomes are
found in the blood. Why?
 Because they failed to be sorted properly in the Golgi apparatus
 So hydrolases are secreted rather than transported to lysosomes
 Miss sorting was occurred due to the defect or missing GlcNAcphosphotranferase – a single gene defect and is like most genetic
enzyme deficiencies, it is recessive
Fig P 610 Alberts 3rd Ed
 Peroxisomes:
 Introduction;
 Peroxisomes differ from mitochondria and chloroplasts in many ways
 Most notably, they are surrounded by only a single membrane and
 Do not contain DNA or ribosomes
 In spite these differences, peroxisomes are thought to acquire their
protein by the similar process of selective import from the cytosol
 Because peroxisomes have no genome therefore all of their proteins must
be imported
 Thus peroxisomes resemble the ER in being self replicating membranebounded organelles that exist without genomes of their own
 Peroxisomes are found in all eukaryotic cells
 They contain oxidative enzymes such as
• catalase and
• urate oxidase at such high concentrations that in some cells of
peroxisomes stand out in electron micrograph
• Because of the presence of a crystalloid core, largely composed of
urate oxidase
Fig 12.27 Alberts 3rd Ed
 Like mitochondrion, peroxisome is a major site of oxygen utilization
 Peroxisomes Use Molecular Oxygen and Hydrogen Peroxide to Carry
out Oxidative Reactions;
 Peroxisomes are so called because they usually contain one or more
enzymes that
 use molecular oxygen to remove hydrogen atoms from specific organic
substrates i.e. R in an oxidative reaction that produces hydrogen peroxide
 Catalase utilizes the H2O2 generated by the other enzymes in the
organelle to oxidize a variety of other substrates including:
• phenols
• formic acids
• formaldehyde and alcohol
by the per oxidative reaction:
• This type of oxidative reaction is particularly important in liver and
kidney cells
• Whose peroxisomes detoxify various toxic molecules that enter the
bloodstream
• About quarter of alcohol people drink is oxidized to acetaldehyde in
this way
• In addition, when excess H2O2 accumulated in the cell, catalase
converts it to H2O
 A major function of the oxidative reactions carried out in peroxisomes is
the breakdown of fatty acid molecules
 In a process called β-oxidation, the alkyl chains of fatty acids are
shortened sequentially by blocks of two carbon atoms at a time
 That are converted to acetyl-CoA and exported from peroxisomes to
cytoplasm for reuse in biosynthetic reactions
 In mammalian cells, β-oxidation occurs both in mitochondria and
peroxisomes
 However, in yeast and plant cells, this essential reaction is exclusively
found in peroxisomes
 An essential biosynthetic function of animal peroxisomes is to catalyze the
first reactions in the formation of plasmalogens - most abundant class of
phospholipids in myelin sheet that insulated the axon of nerve cells
Fig 12.31 Alberts 5th Ed
 Plasmalogen deficiencies cause profound abnormalities in the myelination
of nerve cell axons
 Which is why many peroxisomal disorders lead to neurological disease
 Peroxisomes are unusually diverse organelles and even in the different
cells of a single organism may contain very different sets of enzymes
 They can also adapt remarkably to changing conditions. For example:
• Yeast cells grown on sugar have small peroxisomes but
• When some yeast cells are grown on methanol, they develop large
peroxisomes that oxidize methanol and
• When grown on fatty acids, they develop large peroxisomes that
breakdown fatty acids to acetyl-CoA by β-oxidation
 Peroxisomes also have very important roles in plants
 Two very different types have been studies extensively:
• One type is present in leaves
 where it is catalyzes the oxidation of a side product of the crucial
reaction that fixes CO2 in carbohydrate
Fig 12.28A Alberts 3rd Ed
 This process is called photorespiration because it uses up O2 and
liberates CO2
• The other type of peroxisome is present in germinating seeds and in
filamentous fungi
 Where it plays an essential role in converting the fatty acids stored
in seed lipids in to sugars needed for the growth of the young plant
 Because this conversion of fats to sugars is accomplished by a
series of reactions known as the glyoxylate cycle - a variation of
the tricarboxylic acid cycle and
 these peroxisomes are also called glyoxysomes
Fig 12.28B Alberts 3rd Ed
 In glyoxylate cycle two molecules of acetyl-CoA produced by fatty
acid breakdown in the peroxisome are used to make succinate
 Which leave the peroxisome and converted in to glucose
 The glyoxylate cycle, as mentioned earlier a variation of the
tricarboxylic acid cycle, is an anabolic pathway occurring in
plants, bacteria, protists and fungi
 It does not occur in animal cells and animals are unable to convert
the fatty acids in fats into carbohydrates
Fig down loaded from Web site
 The glyoxylate cycle centers on the conversion of acetyl-CoA to
succinate for the synthesis of carbohydrates
 and allows the conversion of acetyl-CoA to result in net increase in
malate or oxaloacetate, which is not possible with the TCA cycle
alone
 Two acetyl-CoA are input per cycle with no loss of CO2, making
possible net synthesis of a 4-carbon product. The two additional
enzymes of the glyoxylate cycle are isocitrate lyase and malate
synthase
 In microorganisms, the glyoxylate cycle allows cells to utilize
simple carbon compounds as a carbon source when complex
sources such as glucose are not available
1
x
2
x
 A Short Signal Sequence Directs the Import of Proteins into
Peroxisomes;
 A specific sequence of three amino acids (Ser-Lys-Leu) located at the Cterminus of many peroxisomal proteins functions as an import signal
Table 12.3 Alberts 3rd Ed
 Other peroxisomal proteins contain a signal sequence near the Nterminus
 If either sequence attached to a cytosolic protein, the protein is imported
in to peroxisomes
 The import process is still poorly understood, although it is known to
involve both soluble receptor proteins in cytosol
• Which recognize the targeting signals and
• Docking proteins in the cytosol on the cytosolic surface of the
peroxisomes
 At least 23 distinct proteins called peroxines participate in the import
process which is driven by ATP hydrolysis
 A complex of at least six different peroxines forms a membrane
translocator
 Even oligomeric proteins do not have to unfold to be imported in to
peroxisomes, therefore, the mechanism differs from that used by
mitochondria and protoplast
 At least one soluble import receptor, the peroxin - Pex5, accompanies its
cargo all the way into peroxisomes and after cargo release, cycles back to
cytosol
 These aspects of peroxisomal protein import resemble protein transport
in to the nucleus
 The importance of this import process and of peroxisomes is
demonstrated by the inherited human disease Zellweger syndrome
• in which a defect in importing proteins in to peroxisomes lead to a
profound peroxisomal deficiencies
• These individuals, whose cells contain empty peroxisomes have
severe abnormalities in their brain, liver and kidney and they die soon
after birth
•
A mutation in the gen encoding peroxin Pes2, a peroxisomal integral
membrane protein involved in protien import, causes one form of
disease
• Similarly, a defective receptor for N-terminal import signal causes a
milder inherited peroxisomal disease
 It has long been debated whether new peroxisomes arise from preexisting
ones by organelle growth and fission and
 therefore, replicate in an autonomous way like mitochondria and plastids
or they derive as a specialized compartment from the ER
 Aspects of both view may be true
Figure 12.33 Alberts 5th Ed
 Most peroxisomal membrane proteins are made in cytosol and insert in to
the membrane of preexisting ones
 Yet other are first integrated in to the ER membrane from where they
may bud in specialized peroxisomal precursor vesicle
 New precursor vesicles may then fuse with one another and
 begin importing additional peoxisomal proteins using their own protein
import machinery to grow in to mature peroxisomes
 Which can enter in to a cycle of growth and fission
Page 574 Albert 3rd Ed
CELL EXTERIOR
 Mitochondria
 Introduction;
 Mitochondria and chloroplasts are double-membrane enclosed organelles
 They specialize in ATP synthesis using energy derived from electron
transport and oxidative phosphorylation in mitochondria and from
photosynthetic phosphorylation in chloroplasts
 Although both contains its own DNA, ribosomes and other
compartments required for protein, most of their proteins are encoded in
the nucleus and imported from the cytosol
 Each imported protein must reach the particular organelle subcompartment in which it functions
 There are two sub-compartments in mitochondria:
• The internal matrix space and the inter-membrane space
 These compartments are formed by the two concentric mitochondrial
membrane:
• The inner membrane - which encloses the matrix space and forms
extensive invaginations called cristae and
• Outer membrane – which is in contact with cytoplasm
Fig 12.20A Alberts 3rd Ed
 Chloroplasts have the same two sub-compartments plus an additional
sub-compartment – the thylakoid membrane
Fig 12.20B Alberts 3rd Ed
 Each of the sub-compartments contains a distinct set of proteins
 The growth of mitochondria and chloroplasts by import of proteins from
the cytosol is therefore a major feat, requiring
 that proteins be translocated across a number of membranes in
succession and end up in the appropriate place
 The relatively few proteins encoded by the genome of these organelles
are located mostly in the inner membrane in mitochondria and in the
thyakoid membrane in chloroplasts
 These organelle encoded polypeptides generally from subunits of protein
complexes
 Whose other subunits are encoded by nuclear genes and are imported
from the cytosol
 The formation of such hybrid protein complexes requires a balanced
synthesis of the two types of subunits
 How protein synthesis is coordinated on different types of ribosomes
located two membranes apart is still largely a mystery?
 Translocation in to the Mitochondrial Matrix Depends on a
Matrix Targeting Signal;
 Proteins imported in to mitochondria are usually taken up from the
cytosol within seconds or minutes of their release from ribosomes
 Thus mitochondrial proteins are first fully synthesized as mitochondrial
precursor proteins in the cytosol and then translocated in to mitochondria
by a post translational mechanism
 One or more signal sequences direct all mitochondrial precursor proteins
to their approximate mitochondrial sub-compartment
Table 12.3 Alberts 3rd Ed
 Many proteins entering the matrix space contain a signal sequence at Nterminus
• Which is removed by signal peptidase rapidly after import
 Others including outer membrane and many inner membrane and
intermediate space proteins have an internal signal sequence that is not
removed
 The signal sequences are both necessary and sufficient for the import and
correct localization of the proteins
 When genetic engineering techniques are used to link these signals to a
cytosolic protein, the signals direct the protein to the correct
mitochondrial sub-compartment
 The signal sequences that direct precursor proteins in to the
mitochondrial matrix space are best understood

They all form an amphiphilic α helix in which:
• Positively charged residues cluster on one side of the helix
• While uncharged hydrophobic residues cluster on the opposite side
 Specific receptor proteins that initiate protein translocation recognize this
configuration rather than the precise amino acid sequence of the signal
sequence
Fig 12.23 Alberts 5th Ed
Fig 12.21 Alberts 3rd Ed
 Multisubunit protein complexes that function as protein translocators
mediate protein translocation across mitochondrial membranes
 The translocator of outer membrane (TOM) complex transfers proteins
across the outer membrane and two (transporter of inner membrane (TIM
complexes i.e. TIM23 and TIM 22) transfer proteins across the inner
membrane
 These complexes contain some components;
• that act as receptors for mitochondrial precursor proteins and
• other components that form the translocator channels
 The TOM complex is required for the import of all nucleus-encoded
mitochondrial proteins
 It initially transports their signal sequences into the inter-membrane
space and helps to insert transmembrane proteins in to the outer
membrane
 β-barrel proteins – particularly abundant in outer membrane, are then
passed on to an additional translocator, the SAM complex
•
which help them to fold properly in the outer membrane
 The TIM23 complex
• transports some soluble proteins into the matrix space and
• helps to insert transmembrane proteins into the inner membrane
 TIM22 complex mediates the insertion of a subclass of inner membrane
proteins including the transporters that
• moves ADP, ATP and phosphates in and out of mitochondria
 The OXA complex - an other protein translocator located in inner
mitochondrial membrane mediates
• the insertion of those inner membrane proteins that are synthesized
within mitochondria
• It also help to insert some imported inner membrane proteins that are
initially transported into the matrix space by the other complexes
Fig 12.23 Alberts 5th Ed
 The molecular mechanism of protein import into mitochondria has been
done by analyses of
• cell free, reconstituted transport systems in which mitochondria in a
test tube import radiolabel mitochondrial precursor proteins
 Mitochondrial Precursor
Polypeptide Chains;
Proteins
are
Imported
as
Unfolded
 Mitochondrial precursor proteins do not fold in to their native structures
after they are synthesized rather
 They remain unfolded in the cytosol through interactions with other
proteins
 Some of these interacting proteins are:
• general chaperone proteins of the Hsp family whereas
• Others are dedicated to mitochondrial precursor proteins and bind
directly to their signal sequences
 All interacting proteins help to prevent the precursor proteins from
aggregating or folding up spontaneously before they engage with the
TOM complex
• As a first step in the import process, the import receptors of TOM
complex bind the signal sequence of the mitochondrial precursor
protein
•
the interacting proteins are then stripped off and
• unfolded polypeptide chain is fed - first with signal sequence into the
translocation channel
Fig 12.22 Alberts 3rd Ed
Fig 12.25 Alberts 5th Ed
 ATP Hydrolysis and a Membrane Potential Driven Protein Import into
the Matrix Space;
 Directional transport requires energy which in most biological systems is
supplied by ATP hydrolysis
 ATP hydrolysis fuels mitochondrial protein import at two discrete sites
i.e.
i. One outside the mitochondria and
ii. One in the matrix space

In addition to this, protein import requires another energy source
• which is the membrane potential across the inner mitochondrial
membrane
Fig 12.26 Alberts 5th Ed
 Bacteria and Mitochondria use Similar Mechanisms to Insert Porins
into their Membrane;
 Outer mitochondrial membrane, like the outer membrane of Gramnegative bacteria, contains abundant pore forming proteins – porins
Fig 10.18 Alberts 5th Ed
 Thus it is freely permeable to inorganic ions and metabolites but not to
most proteins
 Porins are β-barrel proteins and are first imported through the TOM
complex
Fig 10.26 Alberts 5th Ed
 In contrast to other outer membrane proteins
• Which are anchored in the membrane through α-helical regions, the
TOM complex can not integrate porins into lipid bilayer
 Instead, porins are first transported into inter-membrane space
• where they transiently bind specialized chaperone proteins
• which keep the porins from aggregating
 They then bind to the Sam complex in the outer membrane, which
•
insert them to into the outer membrane and
• help them to fold correctly
Fig 12.27 Alberts 5th Ed
 One of the central subunit of SAM complex is homologous to a bacterial
outer membrane protein that
• helps insert β-barrel proteins in to the bacterial outer membrane from
periplasmic space – the topological equivalent of the inter-membrane
space of mitochondria
• This conversed pathway is further evidence for the endo-symbiotic
origin of mitochondria
 Transport into the Inner Mitochondrial Membrane and Intermembrane Space Occurs via Several Routes;
 The same mechanism that transports proteins into the matrix space, using
the TOM and TIM23 translocators, also mediates the initial translocation
of many proteins
Fig 12.28 Alberts 5th Ed
Page 568 Alberts 3rd Ed
 Function:
 Mitochondria and chloroplasts, membrane bound organelles convert
energy to forms that can be used to drive cellular reactions
 These membrane has a crucial role in the function of this energyconverting organelles by providing a frame work for electron-transport
processes
 Although mitochondria covert energy derived from chemical fuels
whereas chloroplasts convert energy derived from sunlight
 The two types of organelles are organized similarly, moreover, both
produce large amounts of ATP by the same mechanism
 The common pathway by which mitochondria, chloroplasts and even
bacteria harness energy for biological purposes operates by a process
known as chemiosmotic coupling-reflecting a link between the chemical
bond-forming reactions
• That generate ATP (chemi) and membrane-transport processes
(osmotic)
 The energy from oxidation of foodstuffs or from sunlight is used to drive
membrane bound proton pump (H+ pump)
 That transfer H+ from one side of the membrane to the other
 These pumps generate an electrochemical protons gradient across the
membrane
 Which is used to drive various energy-requiring reaction when the
protons flow back “downhill” through membrane-embedded protein
machines
Fig 14.1 Alberts 5th Ed
Fig 14.2 Alberts 5th Ed / Fig 14.1 Alberts 3rd Ed
Fig 14.3 Alberts 5th Ed / Fig 14.2 Alberts 3rd Ed
 The Citric Acid Cycle Generates High-Energy Electrons;
• Mitochondria can use both pyruvate and fatty acids as fuel
• Pyruvate come from glucose and other sugars, whereas fatty acids
come from fats
• Both of these fuel molecules are transported across the inner
mitochondrial membrane and are
• then converted to the crucial metabolic intermediate acetyl CoA by
enzymes located in the mitochondrial matrix
• The acetyl groups in acetyl CoA are then oxidized in the matrix via
the citric acid cycle
• The cycle converts the carbon atoms in acetyl CoA to CO2 which the
cell releases as a water product
• Most importantly, this oxidation generates high-energy electrons
carried by the activated carrier molecules NADH and FADH2
Fig 14.9 Alberts 5th Ed
• These high-energy electrons are than transferred to the inner
mitochondria membrane, where they enter the electron-transport
chain
 The loss of electrons from NADH and FADH2 also generates the
NAD+ and FAD that is needed for continued oxidative
metabolism
Fig 14.10 Alberts 5th Ed
 A Chemiosmotic Process Coverts Oxidation Energy in ATP;
• Although the citric acid cycle is considered to be part of aerobic
metabolism, it does not itself use oxygen
• Only in the final, catabolic reactions that take place on the inner
mitochondrial membrane is molecular oxygen (O2) directly
consumed
• Nearly all energy available from burning carbohydrates, fat and other
food stuffs in the earlier stages of their oxidation initially saved in the
form of high-energy electrons removed from substrates by NAD+ and
FAD
• These electrons carried by NADH and FAD2 then combine with O2
by means of respiratory chain embedded in the inner mitochondrial
membrane harness the large amount of energy released to derive the
conversion of ADP + Pi to ATP
• For this reason, the term oxidative phosphorylation is used to
describe this last series of reactions
Fig14.11 Alberts 5th Ed
 NADH Transfers its Electron to Oxygen Through Three Large
Respiratory Enzyme Complexes;
• Although the respiratory chain harvests energy by a different
mechanism than that used in other catabolic reactions, the principle is
the same
• The energetically favorable reaction H2 + 1/2O2
occur in many small steps
H2O is made to
• So that most of the energy released can be stored instead of being lost
to the environment as heat
• The hydrogen atoms are first separated in to protons and electrons
• The electrons pass through a series of electron carriers in the inner
mitochondrial membrane
• At several steps along the way, protons and electrons are transiently
recombined
• But only at the end of the electron-transport chain are the protons
returned permanently
• When they are used to neutralize the negative charges created by the
final addition of the electrons to oxygen molecule
Fig 14.12 Alberts 5th Ed
 As Electron Move Along the Respiratory Chain, Energy is Stored as
an Electrochemical Proton Gradient Across the Inner Membrane;
• Oxidative phosphorylation is made possible by the close association
of the electron carriers with protein molecules
• The proteins guide the electrons along the respiratory chain so that
the electron move sequentially from one enzyme complex to another
- no short circuits
• Most important, the transfer of electrons is coupled to oriented H+
uptake and release and to allosteric changes in selected protein
molecules
• The net result is that the energetically favorable flow of electron
pumps H+ across the inner membrane, from the matrix space to the
inter membrane space
• The movement of H+ has two major consequences:
 It generates a pH gradient across the inner mitochondrial
membrane, with the higher in the matrix than in the cytosol,
where the pH is generally close to 7
 It generates a voltage gradient (membrane potential) across the
inner mitochondrial membrane with inside negative and out side
positive
 As a result of the net outflow of positive ions
• The pH gradient (ΔpH) drives H+ back in to matrix, thereby
reinforcing the effect of membrane potential (ΔV)
• Which acts to attract any positive ion in to the matrix and to push any
negative ion out
• Together, the ΔpH and the ΔV are said to constitute an
electrochemical proton gradient
• The electrochemical proton gradient exerts a proton-motive force
which can be measured in units of milli volts (mV)
Fig 14.13 Alberts 5th Ed / Fig 14.19 Alberts 3rd Ed
 The Proton Gradient Drives ATP Synthesis;
• The electrochemical proton gradient across the inner mitochondrial
membrane derives ATP synthesis in the critical process of oxidative
phosphorylation
• This is made possible by the membrane-bound enzyme ATP
synthetase - plays a role of a turbine, permitting the protons gradient
to drive the production of ATP
Fig 14.14 Alberts 5th Ed / Fig 14.20 Alberts 3rd Ed
Fig 2.27 Alberts 5th Ed
 The Proton Gradient Drives Coupled Transport Across the Inner
Membrane;
• The electrochemical proton gradient drives other processes besides
ATP synthesis
• In mitochondria many charged small molecules. Such as:
 Pyruvate
 ADP and Pi
are pumped in to the matrix from the cytosol, while others such as:
 ATP must be moved in the opposite direction
NADH dehydrogenase complex
Cytochrome b-c1 complex Cytochrome oxidase
• Transporters that bind these molecules can couple their transport to
the energetically favorable flow of H+ in to mitochondrial matrix
• Thus, for example, pyruvate and inorganic phosphate (Pi) are cotransported in ward with H+ as the H+ moves into the matrix
• ADP and ATP are co-transported in opposite directions by a single
transporter protein
• Since an ATP molecule has one more negative charge than ADP, each
nucleotide exchange results in a total of one negative charge being
moved out of mitochondrion
• Thus the voltage difference across the membrane drives this ADPATP co-tranporter
Fig 14.16 Alberts 5th Ed / Fig 14.21 Alberts 3rd Ed
 Proton Gradients Produce Most of the Cell’s ATP;
• Glycolysis alone produces a net yield of 2 molecules of ATP for
every molecule of glucose that is metabolized and
• This is the total energy yield for the fermentation processes that occur
in the absence of O2
Table 14.1 Alberts 5th Ed
• In conclusion, the vast majority of the ATP produces from the
oxidation of glucose in an animal cells is produced by chemiosmotic
mechanisms in the mitochondrion also produces a large amount of
ATP from the NADH and FAD2 that is derive from the oxidation of
 Proteins can Move Between Compartments in Different Ways;
 The synthesis of all proteins begins on ribosomes in the cytosol
except few that are synthesized on the ribosomes of mitochondria and
plastids
 Their subsequent fate depends on their amino acid sequence, which
can contain sorting signals that direct their delivery to locations
outside the cytosol
 Most proteins do not have a sorting signal and consequently remain in
the cytosol as a permanent residents
 However, many others have specific sorting signals that direct their
transport from the cytosol in to
• the nucleus
• the ER
• mitochondria
• plastids or
• Peroxisomes
 Sorting signals can also direct the transport of proteins from the ER
to other destinations in the cell
 To understand the general principles by which sorting signals operate,
it is important to distinguish
• three fundamentally different ways by which proteins move from
one compartment to another
i. The protein traffic between the cytosol and nucleus occurs
between topologically equivalent spaces, which are in
continuity through the nuclear pore complexes
-
The process is called gated transport because the
nuclear pore complexes function as selective gates
-
that can actively transport specific macromolecules and
macromolecular assemblies
-
although they also allow free diffusion of smaller
molecules
ii. In transmembrane transporter, membrane-bound protein
translocators directly transport specific proteins across a
membrane from the cytosol into a space that is topologically
distinct
-
The transported protein molecule usually must unfold in
order to sneak through the membrane. For example:
 The initial transport of selected proteins from the cytosol
into the
- ER or
- Mitochondria occurs in this way
iii. In vesicular transport, transport vesicles carry proteins from
one compartment to another
 The vesicles become loaded with a cargo of molecules
derived from the luman of one compartment as they pinch
off from its membrane
 They discharge their cargo in to a second compartment by
fusing with its membrane. For example:
-
The transfer of soluble proteins from ER to the
Golgi apparatus occurs in this way
-
Because transported protein do not cross a
membrane, they move only between the
compartments
-
that are topologically equivalent
Fig 12.7 Alberts 5th Ed / Fig 12.6 Alberts 3rd Ed
Fig 12.7 Alberts 3rd Ed / 12.6 Alberts 5th Ed
*
 Each of three modes of protein transfer is usually selectively guided
by sorting signals in the transported protein
 That are recognized by complementary receptor proteins in the target
organelle. For example:
 If a large protein to be imported into the nucleus, it must possess a
sorting signal that is recognized by receptor proteins associated
with the nuclear pore complex
 If a protein to be transferred directly across a membrane, it must
possess a sorting signal that is recognized by the translocator in the
membrane to be crossed
 Similarly, if a protein is to be incorporated into certain types of
transport vesicles or to be retained in certain organelles, its sorting
signal must be recognized by a complementary receptor in the
appropriate membrane
 Signal Peptides and Signal Patches Direct Proteins to the Correct
Cellular Address;
 There are at least two types of sorting signals on proteins
i. This resides in a continuous stretch of amino acid sequence,
typically 15-69 residues long
-
This signal peptide is often but not always removed from
the finished protein by a specialized signal peptidase once
the sorting process has been completed
ii.
This consists of a specific three-dimensional arrangement of
atoms on the protein’s surface that forms when the proteins fold
up
The amino acids residues that comprise this signal patch
can be distant from one another in the linear amino acid
sequences and
they are generally remain in the finished protein
Fig 12.8 Alberts 3rd Ed
Table 12.3 Alberts 3rd Ed
 Cells can not Construct their Membrane-bounded Organelles de
novo: They require Information in the Organelle itself;
 When a cell reproduces by division, it has to duplicate its membranebounded organelles
 In general, cells do this by enlarging the existing organelles by
incorporating new molecules in to them
 The enlarge organelles then divide and are distributed to the two
daughter cells
 Thus each daughter cell inherits from its mother a completer set of
specialized cell membranes
 This heritance is essential because a cell could not make such
membranes de novo i.e. from scratch. For example:
• If the ER were completely removed from a cell, how could the cell
reconstruct it?
• The membrane proteins that define the ER and carry out many of its
functions are themselves products of the ER
• A new ER could not be made without as existing ER or
• At the very least, a membrane that specially contains the translocators
required to import selected proteins in to ER from the cytosol
including the ER specific transporters themselves
• The same is true for mitochondria and plastids
• Thus it seems that the information required to construct an organelle
does not reside exclusively in the DNA that specifies the organelles
proteins
• Information in the form of at least one distinct protein that preexists in
the organelle membrane is also required and
• This information is passed from parent cell to progeny cell in the form
of the organelle itself
• Presumably, such information is essential for the propagation of the
cell’s compartmental organization
• Just as the information in DNA is essential for the propagation of the
cell’s nucleotide and amino acid sequences
 Evolutionary Origins Explain the Topological Relationships of
Organelles;
 To understand the relationships between the compartments of the cell
• It is helpful to consider how they might have evolved?
• The precursors of first eukaryotic cells are thought to have been
simple organisms that resembled bacteria
• Which generally have plasma membrane but no internal
membranes
• The plasma membrane in such cells, therefore provides all
membrane-dependent functions including:
 the pumping of ions
 ATP synthesis
 protein secretion and
 lipid synthesis
• Typical present day eukaryotic cells are 10-30 times larger in
linear dimension and 1000-10,000 times greater in volume than a
typical bacterium such as E. coli
• The evolution of internal membranes evidently accompanied by
specialization of membrane function. For example:
 Consider the generation of thylakoid vesicles in chloroplasts
Fig 12.3 Alberts 5th Ed
• Other compartments in eukaryotic cells may have originated in a
conceptually similar way
• The invagination and pinching off of specialized intracellular
membrane structures from the plasma membrane creates
organelles with an interior that is topologically equivalent to the
exterior of the cell
Fig 12.3 Alberts 5th Ed
Fig 12.4 Alberts 5th Ed
Fig 12.5 Alberts 5th Ed / Fig 12.4 Alberts 3rd Ed
x --------------------------- x---------------------------- x ----------------------------x