THE ENDOMEMBRANE SYSTEM
Endoplasmic Reticulum
• Continuous netwrk of flattened sacs, tubules,
and associated vesicles that stretches through
the cytoplasm
• Endoplasmic— within the (cyto)plasm
• Reticulum— network (Latin)
• ER cisternae— membrane-bounded sacs
• ER lumen— space enclosing cisternae
• Not visible by light microscopy
• Enzymes associated responsible for
o Biosynthesis of proteins destined for
incorporation into the plasma
membrane/organelles
o Synthesis of proteins destined for export
from the cell
• Central role in biosynthesis of lipids including
triacylglycerol, cholesterol, and related
compounds
Two Basic Kinds of Endoplasmic Reticulum
• Rough endoplasmic reticulum
o Ribosomes attached to the cytosilic side
of membrane
▪ Side facing away from ER
lumen
o Translation by ribosomes occur in the
cytosol but newly synthesized proteins
willl enter lumen shortly
o Translational elements (TEs)
▪ Play important role in formation
of transition vesicles
o Transistion vesicles
▪ Shuttle lipids and proteins from
the ER to Golgi
o Usually form large flattened sheets
• Smooth endoplasmic reticulum
o Appears smooth due to absence of
ribosomes
o Form tubular structures
• Rough and smooth ER are not separate
organelles
o Lumenal spaces are continuous
• Cells involved in biosynthesis of secretory
proteins (liver & cells producing digestive
enzymes) have more prominent rough ER
• Cells producing steroid hormones contain
extensive networks of smooth ER
The Rough ER
• Ribosomes on cystolic side, responsible for
synthesizing both membrane-bound and soluble
proteins for endomembrane system
• Synthesis of proteins begin on ribosomes
attached to rough ER via receptor proteins
• Cotranslationally
o How newly synthesized proteins enter
endomembrane system
o Insertion through pore complexes in the
ER membrane into the rough ER lumen
as polypeptide is synthesized by the ER
bound ribosome
• After biosynthesis, membrane spanning
proteins remain anchored to ER via
hydrophobic regions of polypeptide or covalent
attachment to membrane lipid
• Site for other processes such as:
o Initial steps of addition and processing
of carbohydrate groups in glycoprotein
o Folding of polypeptides
o Recognition and removal of misfolded
polypeptides
o Assembly of multimeric proteins
• ER-specific proteins include a host of enzymes
that catalyze cotranslational and
postranslational modifications
• Glycosilation
o Important for sorting proteins into their
proper destinations
• Disulfide bond formation
o Essential for proper protein folding
• ER-asociated degredation (ERAD)
o Proteins improperly modified, folded, or
assembled are exported from the ER
for degradation by cytosolic
proteasomes
The Smooth ER
• Primarily involved in processing or storing
nonprotein molecules within cells
Drug detoxification
• Involves enzyme-catalyzed hydorxylation
o Addition of hydroxyl groups to
hydrophobic drugs
o Make them more soluble and easier to
excrete
• Hydorxylation catalyzed by member of
cytochrome P-450 proteins
o Prevalent in smooth ER of hepatocytes
o Also called monooxygenases
• Reaction (R= drug hydroxylated) see pg. 341 for
full explanation:
 • Tolerance
o Increasing higher doses of drug
necessary to achieve the same sedative
effect
• Synergy
o Combined effect of two substances is
much stronger compared to expected
sum of individual effects
▪ Alcohol + barbiturates, ethanol
inhibits barbiturate hydroxylation
▪ Result: higher than normal
levels of barbiturate
• Aryl hydrocarbon hydroxylase
o Cytochrome P-450 protein
o Metabolizes polycyclic hydrocarbons
▪ Organic molecules composed of
2 or more linked benzene rings
o Increases solubility for such molecules
but oxidized products are often more
toxic than original compound
o Converts some potential carcinogens to
chemically active form
• Pharmacogenetics
o Investigates how inherited differences in
gens (and their resulting protein
products) can lead to differential
responses to drugs and medications
Carbohydrate Metabolism
• Glucose-6-phosphatase
o Membrane-bound enzyme that is
unique to ER
o Hydroluzaes phosphate group from
glucose-6-phosphate to form free
glucose and inorganic phosphate (Pi)
o Reaction:
o Abundant in liver because its major
role is to keep level of glucose in blood
constant
• Glycogen breakdown in liver
o Liver stores glucose as glycogen in
granules associated with smooth ER
o When glucose is needed, glycogen is
broken down phosphorolysis
▪ Produces glucose-1-
phosphate
o Glucose-1-phosphat converted to
glucose-6-phosphate by
phosphoglucomutase
o Glucose-6-phosphate converted to
free glucose by glucose-6-
phosphatase
o Free glucose leaves liver cell via
GLUT2 and moves into the blood for
transport to cells that need energy
• Significant glucose-6-phosphatase activity in
liver, kidney, and intestinal cells
• Muscle and brain cells retain glucose-6-
phosphate and use it to meet their own energy
needs
Calcium Storage
• Sarcoplasmic reticulum— specialized smooth
ER that store Ca in myocytes
• In these cells, ER lumen contains high conc. Of
Ca-binding proteins
• ATP-dependent calcium ATPases
o Pumps calcium ions into the ER
• Calcium ions released in response to
extracellular signals to aid muscle contraction
o Binding of neurotransmitter molecules to
receptors on cell surface
Steroid Biosynthesis
• Smooth ER in certain cells is the site of
biosynthesis of cholesterol and steroid
hormones (cortisol, testosterone, and estrogen)
• Large amounts of smooth ER in:
o cortisol-producing cells of adrenal gland
o Leydig cells of testes (testosterone)
o Follicular cells of ovary (estrogen)
• Smooth ER also found in plastids of some plants
where it may be involved in plant hormone
synthesis
• Hydroxymethylglutaryl-CoA reductase (HMG-
CoA reductase)
o The committed step in cholesterol
biosynthesis
o Targeted by statins
▪ Cholesterol-lowering drugs
• P-450 monooxygenases
o Important in synthesis of cholesterol as
well as its conversion into steroid
hormones by hydroxylation
Role of ER in Membrane Biosynthesis
• ER is the primary source of membrane lipids
including phospholipids and cholesterol
• Exceptions:
o Mitochondria synthesize
phosphatidylethanolamine by
 decarboxylation great imported
phosphatidylserine
o Peroxisomes have enzymes ito
synthesize cholesterol
o Chloroplasts contain enzymes for
synthesis for chloroplasts-specific lipds
• Biosynthesis of fatty acids for membrane
phospholipids occurs in the cytoplasm
• Incorporation is restricted to the monolayer of
the ER membrane facing the cytosol
• Flippases
o Phospholipid translocations
o Catalyze translocation of phospholipids
through ER membranes
o Specific and affect only the rate of a
process
• Type of phospholipids molecules transferred
across a membrane depends on the
translocators present
o ER membrane contains trans locator for
phosphatidylcholine, thus it is found in
both monolayers of ER
o But no translocators for
phosphatidylethanolamine/linositol/seri-
ne and are therefore confined in
cystosolic monolayer
• Phospholipids exchange/transfer proteins
o Convey phospholipid molecules from ER
membrane to outer mitochondrial and
choloroplast membranes
o Recognizes specific phospholipid,
removes it from one membrane and
carries it through the cytosol of another
membrane
• Although ER is the source of most membrane
lipids, its composition varies from other cellular
membranes
Golgi Apparatus
• Closely linked physically and functionally to the
ER
• Name derived from Camillo Golgi, the Italian
biologist first to describe it
• A series of flattened membrane-bounded
cisternae
o Disk-shaped sacs that are stacked
together
o Usually 3-8 cisternae per stack
• Number and size of Golgi stacks vary with the
cell type and with metabolic activity of the cell
• Both ER and Golgi are dynamic structures
o Typically surrounded by numerous
vesicles that carry lipids and proteins
form the ER to the Golgi to other
organelles
• Lumen
o Intracisternal space of the golgi
• Cis-face
o oriented toward the ER
o cis-Golgi network (CGN)
▪ A network of flanttened,
membrane-bounded tubules
▪ Golgi compartment closes to the
ER
o Vesicles containing newly synthesized
lipids and proteins form the ER
continuously arrive at the CGN where
they fuse with CGN membranes
• Trans-face
o Opposite side of the Golgi, oriented
towards the cytosol
o trans-Golgi network (TGN)
▪ similar morphology to CGN
o Proteins and lipids leave the golgi via
transport vesicles that continuously bud
form the tips of TGN cisternae
• Medial cisternae
o Central stacks between CGN and TGN
o Where much of the processing of
proteins occur
• CGN, TGN, and medial cisternae are
biochemically and functionally distinct
o Each compartment with specific receptor
proteins and enzymes
Models That Account for the Flow of Lipids and
Proteins through the Golgi
• Stationary Cisternae Model
o Each compartment of the golgi stack is a
stable structure
o Trafficked between successive cisternae
is mediated by shuttle vesicles
▪ Bud from one cisterna and fuse
with the next cisterna in a cis-to-
trans sequence
o Protein destined for TGN are carried
forward by shuttle. Vesicles
o Molecules that belong in the ER and
successive Golgi compartments are
actively retained for retrieved
• Cisternal Maturation Model
Role of ER and Golgi in Protein Glycosylation
o Golgi cisternae are transient
compartments that gradually change
from CGN cisternae through medial
cisternae to TGN cisternae
o Transition vesicles from the ER
converge to from the CGN
▪ Accumulates specific enzymes
for early steps of protein
processing
o Each cis cisterna is transformed first into
an intermediate medial cisterna and
then into a trans cisterna as it acquires
additional enzymes
o Enzymes no longer needed in later
compartments return in vesicles to early
compartments
• In both models TGN forms transport vesicles or
secretory granules containing sorted cargo
targeted for various destinations
• Both may apply to some degree, depending on
the organism and role of the cell
• Anterograde transport
o Antero— front, Latin
o Movement of material from the ER
through the Golgi towards the plasma
membrane
o Secretory granule fuses with the plasma
membrane and discharges its contents
(exocytosis), a bit of membrane from ER
becomes part of the plasma membrane
• Retrograde transport
o Retro— back, Latin
o Flow of vesicles form Golgi cisternae
back to ER
o Balance the flow of lipids toward the
plasma membrane
o Ensure a supply of components for
forming new vesicles
▪ Cell recycles lipids and proteins
no longer needed in late stages
of anterograde
• In stationary cisternae model, retrograde flow
facilitates:
o The recovery of ER-specific lipids and
proteins passed from ER to CGN
o Transport of compartment-specific
proteins back to distinc medial cisternae
• Material for TGN continues forward
• In the cisternal maturation model, retrograde
flow carries material back to newly forming
compartments
o When receptors and enzymes no longer
needed by more mature compartments
• Glycosylation
o Addition of carbohydrate side chains to
specific amino acid residues of proteins
forming glycoproteins
• N-linked Glycosylation
o Involve the addition of a specific
oligosaccharide unit to the nitrogen atom
of a terminal amino group of certain
asparagine residues
• O-linked Glycosylation
o Involves additio of an oligosaccharide to
the oxygen atom on the hydroxyl group
of certainty serin e or threonine residue
Initial Glycosylation Occurs in the ER
• Specific enzymes that catalyzes various steps of
Glycosylation and modifications are present in
specific compartments of the ER and Golgi
• Initial steps of N-Glycosylation takes place on
the cytosolic surface of the ER membrane
• Late steps occur in the ER lumen
• Core oligosaccharide
o Two units of N-acetylglucosamine
(GlcNAc), 9 mannose units, and 3
glucose units
o Initial makeup of all carbohydrate side
chains added to the ER
• Glycosylation begins as dolichol phosphate, an
oligosaccharide carrier, is inserted into ER
membrane
• GlcNAc and mannose groups are then added to
the phosphate group of dolichol phosphate
• The growing core oligosaccharide is then
translocated from the cytosol to the ER lumen by
a flippase
• Once inside the ER lumen, more mannose and
glucose units are added
• The completed core oligosaccharide is then
transferred as a single unit from dolichol to an
asparagine residue of the recipient protein
• The core oligosaccharide attached to the protein
is trimmed and modified
• Cotranslational Glycosylation
o Core oligosaccharide is added to the
protein as polypeptide is being
synthesized
o Helps promote proper protein folding
• Experimental inhibition of glycosylation leads to
appearance of misfolded, aggregated proteins
• Calnexin (membrane-bound) and Calreticulin
(soluble)
o ER proteins
o Either can bind to the monoglucosylated
glycoproteins and promote proper
folding
▪ Form a complex with the
glycoprotein and Erp57
• A thiol oxidoredictase
• Catalyzes disulfide
bond formation
o Protein complex then dissociates and
final glucose unit is removed by
glucosidase II
• UGGT (UDP-glucose: glycoprotein
glucotransferase)
o A specific glucosyl transferase in ER
o Acts as a sensor for proper folding of the
newly synthesized glycoprotein
o Binds to improperly folded proteins and
adds back a single glucose unit
▪ Makes the protein a susbstrate
for another round of
calnexin/calreticulin binding and
disulfide bond formation
o Once proper conformation is achieved,
UGGT is no longer bound to the
glycoprotein allowing it to move to the
Golgi
Further Glycosylation Occurs in the Golgi
• Terminal glycosylations
o Further processing of the N-
glycosylated proteins occurs as it moves
though the compartments (cis to medial
to trans)
o Show variability among proteins and
account for much of the great diversity in
structure and functio of protein
oligosaccharide side chains
o Includes removal of a few carbohydrate
units of the core oligosaccharides
• Some case no further prodcessing occurs in the
Golgi
• Others, more complex oligosaccharides, are • For anterograde
generated by further addition og GlcNAc and o (1) As ribosomes of the rough ER
other monosaccharides including galacoe and synthesize proteins, proteins enter the
sialic acis ER lumen where intial glycosylation
• Glucan synthesases occurs
o Enzyme class in golgi stacks o (2) Transition vesicles carry
o Produce oligosaccharides from Glycosylated proteins and newly
monosaccharides synthesized lipids to the CGN
• Glycosyl transferases o (3) Lipids and proteins move through the
o Enzyme class in golgi stacks cisternae of the Golgi stack
o Attach carbohydrate groups to proteins o (4a) At the TGN, some vesicles form
o Also found in the ER secretory vesicles, which release their
contents at the plasma membrane by
Roles of the ER and Golgi in Protein Trafficking exocytosis
• Each protein contains a specific “tag” targeting o (4b) Other vesicles bud from the TGN to
the protein to a transport vesicle that will carry form endospores that help make
material from one specific cellular location to lysosomes
another • For retrograde
• Depending on the protein and its destination the o (1) At the same time, proteins and other
tag may be: materials can be taken in to the cell by
o A shower amino acid sequence endocytosis, forming vesicles that fuse
o An oligosaccharide side chain with early endosomes
o A hydrophobic domain o (2) Early endosomes containing this
o Other structural features material for digestion mature to form late
endosomes and then lysosomes
• Tags may also be involved in excluding material
o (3) Retrograde traffic returns
from certain vesicles
compartment-specific proteins to earlier
• Membrane lipids may also be tagged to hel
compartments
vesicle reach their proper destinations
• Tags for membrane lipids may be one or more ER-Specific Proteins Contain Retention and Retrieval
phosphat groups attached to positions 3,4,
Tags
and/or 5 of a membrane phosphatidylinositol (PI)
• Protein composition in ER maintained by
molecule by a specific kinase
preventing some proteins from escaping and by
• Aside from specific tags, length and degree of retrieving other proteins that have left the ER
saturation of lipids are also important in vesicle and reached CGN
trafficiking
• Several proteins in ER contain the tripeptide
• Sorting newly synthesized proteins begins in the sequence RXR (Arg-X-Arg, X any amino acid)
ER and early golgi compartments o Promotes retention in the ER
o Contain mechanisms for retrieving or
• This retention tag also found in some
retaining compartment-specific proteins
multisubunit proteins destined for plasma
to maintain integrity of glycosylation and
membrane
process pathways
• Many soluble ER-specific proteins contain
• Final sorting occurs in the TGN where lipids and
retrieval tags that bind to specific
proteins are selectively packaged into transport
transmembrane receptors facing the Golgi
vesicles
lumen
• Golgi can also be invovled in the processing of
• Tags are short C-terminal amino acid sequences
proteins that enter the cell by endocytosis
such as:
• o KDEL (Lys-Asp-Glu-Leu)
 o KKXX (X can be any amino acid) in
mammals
o HDEL (His-Asp-Glu-Leu) in yeast
• When protein containing this tag binds to
receptor
o Receptor undergoes a conformational
change
o Then receptor-ligand complex is
packaged into a transport vesicle for
return to ER
• Chimeric/fusion proteins
o Used in experiments for evidence of
importance of retrieval tags
o Made by joining the DNA sequences
encoding 2 polypeptide segments from
diff. proteins (hybrid polypeptide)
Golgi Proteins may be Sorted According to the Lenths of
Their Membrane-Spanning Domains
• Resident proteins of the Golgi also contain
retention or retrieval tag
• In the Golgi, the formation of large complexes
that are excluded from transport vesicles may
play a role in maintaining protein composition
• All known Golgi-specific proteins are integral
membrane proteins hat have one or more
hydrophobic membrane-spanning domains
anchoring them
• Length of hydrophobic domains may determine
into whichh cisterna of the Golgi it is
incorporated in as it moves through the
organelle
• There is a corresponding increase in the lengths
of hydrophobic membrane-spanning domains
going from the CGN to the TGN
• Proteins tend to move from compartment to
compartment until thickness of their membrane
exceeds the length of their membrane-spanning
domain
o Blocks further migration
Targeting Soluble Lysosomal Proteins to Endosomes
and Lysosomes is a model for Protein Sorting in TGN
• In the Golgi, mannose residues on the
carbohydrate side chain of lysosomal enzymes
are phosphorylated
o Forms oligosaccharide containing
mannose-6-phosphate
• This oligosaccharide tag distiguishes soluble
lysosomal proteins from other glycoproteins
o Ensures that they are delivered to the
lysosomes
• (1) In the ER, soluble lysosomal enzymes
undergo N-glycosylation followed by removal of
glucose and mannose units
• (2) In the Golgi, mannose residues are
phosphorylated by two golgi-specific enzymes
o Phosphotransferase— Adds GlcNAc-1-
phosphate to carbon atom 6 of mannose
▪ Found in an early compartment
of the Golgi stack
o The second enzyme removes GlcNAc
leaving behind mannose-6-phosphate
▪ Found in mid-Golgi
compartment
• (3) In the TGN, tagged lysosomal enzymes bind
to mannose-6-phosphate receptors (MPRs) in
the membrane
o pH 6.4 of TGN favors binding of soluble
lysosomal enzymes to these receptors
o Receptor-ligand complexes are packed
into transport vesicles and conveyed an
endosome
• Late endosomes
o Where lysosomal enzymes needed for
degradation of material brought in via
endocytosis is delivered to from TGN
• Early endosomes
o Precursor of late endosomes
o Formed by coalescence of vesicles from
TGN and plasma membrane
• Multivesicular endosomes (MVEs) or
Multivescular bodies (MVBs)
o Specialized endosome
o Carries out degradation and recycling of
unneeded or damaged cell components
o Serve as intermediate between early
endosomes and lysosomes
 o Contain intraluminal vesicles that form
by budding into the interior of the MVEs
o Can later fuse with a lysosome to
degrade its contents or material in the
MVE
• (4) Bound lusosomal enzymes dissociate from
MPRs
o Due to decrease of lumen pH to 5.5
o Happens as early endosome mature to
late endosomes
o Prevents retrograde movement as the
receptors are recycled in vesicles
returning back to TGN
o Late endosome either matures to form a
new lysosome or delivers its content to
an active lysosome
Exocytosis and Endocytosis: Transporting Material
Across the Plasma Membrane
• Exocytosis
o Process by which secretory granules
release their contents to the exterior of
the cell
• Endocytosis
o Process by which cells internalize
external materials
• Both process unique to eukaryotic cells
• Also invovled in delivery, recycling, and turnover
of membrane proteins
Secretory Pathways Transport Moleciles to the Exterior
of the Cell
• Secretory pathways
o Pathways by which proteins move from
the ER through the Golgi to secretory
vesicles and granules
• Secretory vesicles and granules then discharge
their contents to the exterior of the cell
• James Jamieson and George Palade (1967)
demonstrated the concerted roles of the ER and
Golgi in secretion
o Used secretory cells from guinea pig
pancreatic slices
o Results:
• Based on this experiment and similar studies,
the secretory pathways are now understood in
considerable detail
• Randy Schekman and colleagues
o Identified an number of Sec proteins
required at various stages of the
secretion process
o We named as such due to mutations in
genes that encode them
▪ Led to defective secretion of
glycoprotein
• Constitutive secretion
o Involves the continuous discharge of
vesicles at the plasma membrane
surface
• Regulated secretion
o Involves controlled, rapid releases that
happen in response to the extracellular
signal
• Polarized secretion
o Involves secretion at one specific end of
the cell
Constitutive Secretion
• After budding form the TGN some secretory
vesicles move directly to the cell surface
• There, they immediately fuse with the plasma
membrane and release contents via exocytosis
• An unregulated process, which is continuous
and independent of specific extracellular signals
• Ex. continuous release of mucus by cells that
line the intestines
• Once assumed to be a default pathway for
proteins synthesize by the rough ER
o All proteins are destined to remain in the
endomembrane system must have a tag
that diverts the from constitutive
secretion
o Otherwise, they will move through that
endomembrane system and be released
outside the cells
• However, more recent evidence suggest that a
variety of short amino acid tags may identify
specific proteins for constitutive secretion
Regulated Secretion
• Secretory vesicles accumulate in the cell and
then fuse with the plasma membrane only in
response to extracellular signals
• Ex. release of neurotransmitters, release of
insulin, and release of zymogens (inactive
precursors of hydrolitic enzymes)
• Regulated secretory vesicles form by budding
from the TGN as immature secretory vesicles
• They then undergo a maturation process
o Involves concentration of the proteins—
condensation— and frequently some
proteolytic processing
• Mature secretory vesicles are the moved close
to the site of secretion and remain near the
plasma membrane until receiving a signal
o Rejiggers release of contents by fusion
with plasma membrane

• Zymogen granules
o Type of mature regulated secretory
vesicle
o Usually quite large and contain highly
concentrated protein
o Concentrated in the region between
Golgi stacks and portion of the plasma
membrane bordering the lumen
• Information needed to direct a protein in this
secretion is presumably inherent in the amino
acid sequence
• It is sugggested that high conc. of secretory
proteins in secretory granules promote formation
of large protein aggregates
o Excludes non secretory proteins
o Can occur in TGN or in the secretory
granule itself
o pH of TGN and secretory granule may
serves as a trigger for aggregation
o Proteins that did not aggregrate would
then be carried out by transport vesicles
Polarized secretion
• Secretion of specific proteins is limited to a
specific surface of the cell
• Ex. secretory cells lining the intestine release
digestive enzymes only on the side of the cell
that faces the interior of the intestine
• Proteins and lipids are sorted into vesicles that
bind to localized recognition sites on sub
domains of the plasma membrane
Exocytosis Releases Intracellular Molecules Outside the
Cell
• Proteins or other materials in a vesicle are
release to the exterior of the cell as the
membrane of the vesicle fuses with the plasma
membrane
• Animal cell secrete peptide and protein
hormones, mucus, and several digestive
enzymes in this manner
• Plant and fungal cells secret enzymes and
structural proteins associated with the cell wall
and carnivorous plants secrete hydrolitic
enzymes to digest trapped insects
• (1) Vesicles containing cellular products
destined for secretion move to the cell surface
• (2) Membrane of the vesicle fuses with the
plasma membrane
• (3) Fusion with the plasma membrane
discharges the vesicle contents to the exterior of
the cell
• (4) In the process, the membrane of the vesicle
becomes integrated into the plasma membrane
o The inner (lumenal) surface of the
vesicle becomes the outer (extracellular)
surface of the plasma membrane
• Evidence points to the involvement of
microtubules in vesicle movement toward the
plasma membrane
Role of Calcium in Trigerring Exocytosis
• Fusion of regulated secretory vesicles with the
plasma membrane is generally trigerred by a
specific extracllular signal
o Most cases a hormone or
neurotransmitter
• During regulated secretion, a transient elevation
of the intracellular concentration of calcium ions
o Appears to be essential in signaling
cascade leading from receptor on the
cell surface to exocytosis
• Ex. micro injection of Ca into pancreatic cells
induces mature secretory granules to discharge
contents
 • Specific role of Ca remains unclear Phagocytosis
• But elevation of its intracellular conc. Leads to
the activation of protein kinases
o Targets proteins that are components of
the vesicle membrane or plasma
membrane
Endocytosis Imports Extracellular Molecules by Forming
Vessicles From the Plasma membrane
• Greek: cellular eating
• Ingestion of large particles (>0.5 µm diameter),
including:
o aggregates of macromolecules
o parts of other cells
o Whole microorganisms
o Other cells
• For many unicellular eukaryotes, it is a routine
means of acquiring food
• In some primitive animals, notably flatworms,
coelenterates, and sponges, it a means of
obtaining nutrients
• In complex organisms it is restricted to
phagocytes
o Specialized cells that perform
phagocytosis
• Examples
o Neutrophils—engulf and digest foreign
material or invasive microorganisms
o Macrophages— similar with neutrophils
with the addition of ingesting cellular
debris and whole damaged cells
o Fibroblasts—take up collages to allow
remodeling of tissue
o Dendritic cells (mammalian spleen)
ingest bacteria
• Pseudopods

• (1) A small segment of the plasma membrane

progressively forms inward
• (2-4) It then pinches off to form an end cystic
vesicle contains ingested substance or particles
• Endocytosis is important in processes such as:
o Ingestion of essential nutrients by some
unicellular organism
o Defense against microorganisms by
white blood cells
• Endocytosis removes lipids and proteins from
the plasma membrane
• Through endocytosis and retrograde transport,
the cell can recycle and reuse molecules
deposited in the plasma membrane by secretory
vesicles during exocytosis
• The membrane of an enocytic vesicle isolates
the internalized substances from the cytosol
• Most endocytosis vesicles develop into early
endosomes, which fuse with vesicles from TGN
o Acquires digestive enzymes and
matures to form new lysosomes
• Phagocytosis
o Greek: “cell eating”
o Large solid particles are ingested
• Pinocytosis
o Greek: “cell drinking” o Folds of a membrane usually found in
o Liquids containing soluble or suspend smaller organisms (e.g. bacteria)
molecules are taken up o Surrounds the object and meat and
engulf the particle forming an
intracellular phagocytic vacuole
 o Endocytic vesicle or phagosome then
fuses with a late endosome or matures
into a lysosome
• Human phagocytes generate toxic conc. of
hydrogen peroxide, hypochlorous acid and other
oxidants in the phagocytic vacuole to kill
microorganisms
Receptor-Mediated Endocytosis
• Use specific receptors on outer surface of
plasma membrane to acquire soluble and
suspend materials
• Primary mechanism form the specific
internalization of most macromolecules by
eukaryotic cells
• Many kinds of macromolecules taken up, each
having their own specific receptor
• (2) Coated pits
o Sites of collection and internalization of
receptor-ligand complexes
o 20% of total surface area of plasma
membrane (mammals)
• (3) Accumulation of receptor-ligand complexes
in coated pits trigger accumulation of additional
proteins in cytosilic surface of plasma
membrane
o Includes clathrin, clathrin adaptor
proteins and dynamic
o Required to promot membrane
curvature and invagination of the pit
• (6) Coat proteins and dynamic recycle to the
plasma membrane where they become
available for forming new vesicles
o Uncoated vesicle free to fuse with early
endosome
• A coated pit usually invaginates within a minute
or so of being formed
• In fibroblast cells up to 2,500 coated pits are
invaginated per minute
• Progressive formation of a coated vesicle from
a coated pit:
• Epidermal Growth Factor (EGF)
o Stimulated division of epithelial cells
o Undergoes endocytosis by mechanism
in figure 12-15
o As EGF receptors are internalized, the
cell becomes less responsive to EGF
(desentization)
o Deficiency in desentization caused by
defective endocytosis can lead to
▪ Excessive cell growth
▪ Excessive cell division
▪ Possible tumor formation
• One variation has receptors concentrated in
coated pits independent of formation of
receptor-ligand complexes
o Binding of ligands of receptors simply
triggers internalization
• In another variation, receptors are constituvely
concentrated and constitutive internalized
regardless of whether ligands have bound to
receptors
• After receptor mediated-endocytosis, uncoated
vesicles fuse with vesicles form TGN to form
early endosomes
• Early endosomes
o Sites for sorting and recycling
extracellular material brought into the
cell by endocytosis
• As early endosomes continue to acquire
lysosomal proteins in TGN it matures into a late
endosome which develops into a lysosome
• Recycling of plasma membrane receptor
molecules is facilitated by acidification of early
endosome
• ATP-dependent proton pump
o Maintenance the low pH in the
endosomes membrane
• Slightly acid is enviroment of early endosome
decrease the affinity of most receptor ligand
complexes
o Frees receptors to be recycled to the
plasma membrane
o While newly ingested material is
diverted to other locations
 • Depending on the ligand, some receptor-ligand
complexes don’t dissociate in the early
endosome
• Intact receptor-ligand complexes are subject to
sortin and packaging into transport vesicles
• Alternative fates for these complexes
o Carried to a lysosome for degredation
(e.g. epidermal growth factor and its
receptor)
o Carried to the TGN, where they enter a
variety of pathways transporting
material through the endomembrane
system
o Travel by transport vesicles to a
different region of the plasma
membrane where they are secured as a
part of transcytosis
Clathrin-Independent Endocytosis
• Fluid-phase endocytosis
o A type of pinocytosis for non-specific
internalization of extracellular fluid
o Does not concentrate ingested material
o Concentration of the material trapped in
vesicles reflects its concentration in the
extracellular environment
o Proceeds at a relatively constant rate in
most eukaryotic cells
o A means of controlling a cell’s volume
and surface area
o Once inside the cell, fluid-phase
endocytic vesicles are routed to early
endosomes
Coated Vesicles in Cellular Transport Processes
• Vesicles invovled in lipid and protein transfer
• Have characteristic coats, or layers, of proteins
covering their cytosolic surfaces
• Thomas Roth and Keith Porter (1964)
o First to describe coated vesicles
o Involvement in uptake of yolk protein of
mosquito oocytes
• Involve in vesicular traffic and transport during
exocytosis and endocytosis
• Clarithin, COPI and COPII(COP abbreviation for
coat protein) are the most studied coat protein
• Coat proteins participate in formation of
transport vesicles
• Other roles include
o Forcing nearly flat membranes to form
spherical vesicles
o Prevent premature, nonspecific fusion of
a budding vesicle with nearby
membranes
o Regulation interactions between
budding vesicles and mircotubles
• Clarithin-coated vesicles
o Involved in selective transport of
proteins from the TGN to endosomes
o Endocytosis of receptor-ligand
complexes from the plasma membrane
• COPI-coated vesicles
o Facilitate retrograde transport of
proteins from Golgi bacteria to ER as
well as between cisternae of Golgi
• COPII-coated vesicles
o Involved in the transport of material from
ER to Golgi
• Caveolin-coated vesicles
o Caveolae (Latin: “little caves”)
▪ Small, flask-shaped
invaginations of plasma
membrane
▪ Characterized by presence of
cholesterol-binding protein
caveolin
o May be involved in cholesterol uptake by
cells
o Other proposed roles
▪ Participation in endocytosis and
exocytosis
▪ Redox sensing and signal
transduction
▪ Regulation of airway function in
lungs
o Caveolae have been shown to contain
proteins importan in calcium signalling in
heart muscles
Clarithin-Coated Vesicles
• Composed of two multimeric proteins, clathrin
and an adaptor protein complex (AP)
• Clathrin
o From clathratus, Latin: “lattice”
• Clathrin and AP assemble to form protein
lattices composed of polygons
o Flat clathrin lattices— hexagons
o Curved lattices— hexagons and
pentagons
▪ Form under coated pits and
surround vesicles

• Ernst Ungewickell and Daniel Brandon (1981)
o Visualized the basic structural units of
clathrin lattices, three legged structures
called triskelions
• Triskelion
o Multimeric protein composed of:
▪ Three large polypeptides (heavy
chains)
▪ Three smal poly peptides (light
chains)
• Eukaryotic cells contain at least 4 types of AP
complexes each composed of:
o Four polypeptides
▪ Two adaption subunits
▪ One medium chain
▪ One small chain
o Slightly different in each type of AP
complex
• Function of AP complexes
o Bind to diff. transmembrane receptor
proteins
o Confer specificity during vesicle
budiding and targeting
o Ensure appropriate macromolecules will
concentrated in coated pits
o Mediate tha attachment of clathrin to
proteins embedded in the plasma
membrane
o Regulation for clathrin assembly and
disassembly
• Dynamin
o Cytosilic GTPas required for coated pit
constriction and closing of budding
vesicle
o Participates in vesicle as clathrin
accummulates around budding vesicle
• As GTP is hydrolyzed,dynamin rings tighten and
separate the fully sealed endocytic vesicle
• Dissociation/uncoating of clathrin coat is an
energy consuming process
o Hydrolysis of 3 ATP molecules per
triskelion
o Uncoating ATPase
▪ Releases clathrin triskelinons
from AP
COPI- and COPII- Coated Vesicles
• COPI-coated vesicles
o Found in all eukaryotic cells
o Involved in retrograde transport from
Golgi to ER
o Also invovled in bidirectional transport
between Golgi cisternae
• Coat surround the COPI vesicle made up of:
o COPI protein
▪ Multimer w/ 7 subunits
o ADP ribosyl factor (ARF)
▪ Small GTP-binding protein
▪ Mediates assembly of COPI
coat
• Brefeldin A
o Interferes with the ability of guanine
nucleotide exchange factor to produce
ARF-GTP from ARF-GDP
• COPII-coated vesicles
o First discovered in yeast where they
have a role in transport form ER to
Golgi
o COPII coats in yeast are made up of
▪ 2 proten complexes
• Sec13/31
• Sec 23/24
▪ SarI
• Small GTP-binding
protein
SNARE Proteins

Clathrin Coats and Formation of Vesicles in Plasma

Membrane and TGN
• Assembly of clathrin coats on cystolic side
• SNARE hypothesis
provide part of the driving force for vesicle
o Proposed by James Rotham colleagues
formation
 o Provides a working model for important
sorting and targeting step in intracellular
transport
• Identified 2 proteins necessary for secretion
o N-ethylamaleimide-sensitive factor
(NSF)
o Soluble NSF attachment proteins
(SNAPS)
• Accdng. to the hypothesis the proper sorting and
targeting of vesicles in eukaryotic cells involve 2
families of SNARE (SNAP) proteins
o v-SNAREs (vesicle-SNAP proteins)
▪ found on transport vesicles
o t-SNARE proteins (target-SNAP
receptors)
▪ found on target membranes
• v-SNAREs and t-SNAREs are complementary
molecules that allow a vesicle to recognize and
fuse with target membrane
• Rab GTPases
o Vesicles fated for diff. destinations have
distinct members of the Rab family
associated with them
• After initial contact, rapid “zippering” together of
the v- and t-SNARE proteins provides force
necessary to drive membrane fusion
• After vesicle fusion, NSF and group SNAPs
mediate release of v- and t-SNAREs of donor
and targe membranes
• Tethering proteins
o Acts over long distances
o Provides specificity by connecting
vesicles to their target membranes
▪ Prior to v-SNARE/t-SNARE
interaction
• Golgins
o First class of tethering proteins
o Coiled-coil proteins
o Important in initial recognition and
binding of COPI- or COPII-coated
vesicles to Golgi
o Also important in connecting Golgi
cisternae to one another
• Second class of tethering proteins is
multisubunit protein complexes containing 4-8 or
more individual proteins
Lysosomes
• Organelle in the endomembrane system
containing enzymes capable of degrading all
major classes of biological macromolecules
o Lipids, carbohydrates, nucleic acids, and
proteins
• Hydrolitic enzymes
o Degrade extracellular materials brought
into the cell by endocytosis
o Digest intracellular structures and
macromolecules that are damaged or no
longer needed
• First discovered by Christian Duve and
colleagues (1950s)
o Found that acid phosphatases and
several other hydrolitic enzymes were
associated to the lysosome
• Characteristics of lysosomes
o Generally 0.5 µm in diameter
o Bounded by a single membrane
▪ Protects the rest of the cell from
hydrolitic enzymes in lysosomal
lumen
o Acidic environment (pH 4.0-5.0)
▪ Maintained by ATP-dependent
proton pumps
• Favors enzymatic digestion of macromolecules
by:
o Activating acid hydrolysis
o Partially denaturing macromolecule
targeted for degradation
• Products of digestion transported to the cytosol
where they either:
o Enter various synthetic pathways
o Exported from the cell
• Acid hydrolysis
o Common property of lysosomal
enzymes
o Hydrolitic enzymes with pH optimum at
5.0
o 5 phosphatases, 14 proteases and
peptidases, 2 nucleases, 6 lipases, 13
glycosidases, and 7 sulfatases
Lysosomes Develop from Endosomes
• Lysosomal enzymes are synthesized by
ribosomes at the rough ER
• They are then translocated through a pore in
the ER membrane into the ER lumen before
transport to Golgi
• After modification and processing in ER and
Golgi, they are sorted from other proteins in
TGN
• They are then packed into clathrin-coated
vesicles, lose their protein coate and travel to
one of the endosomes compartments
• Last step in lysosome development is the
activation of acid hydrolysases
Lysosomes Enzymes and Different Digestive Processes
• Site of activity is intracellular
• Some cases, lysosome may release enzymes
outside cel via exocytosis
• Heterophagic lysosomes
o Contain substances of extracellular
origin
• Autophagic lysosomes
o Contain materials of intracellular origin
• Specific process where lysosomal enzymes are
involved
o Phagocytosis
o Receptor-mediated endocytosis
o Autophagy
o Extracellular digestion
o See figure 12-21

Phagocytosis and Receptor Mediated Endocytosis:
Lysosomes in Defense and Nutrition
• Degredation of foreign material brought into
eukaryotic cells
• Phagocytic vacuoles are transformed into
lysosomes by fusing with early endosomes (A)
• Vesicles containing material brought into a cell
by receptor mediated endocytosis form early
endosomes (B)
• As early endosomes fuse with TGN w/ acid
hydrolysis the mature into late endosomes and
lysosomes
o Where ingested material is digested
• Residual body
o Idigestible material that remain in the
lysosome
Autophagy: A Biological Recycling System
• Lysosomes break down cellular structures and
components that are damaged or no longer
needed
• Autophay is the digestion of old or unwanted
organelles or other cell structures
o Greek: “self-eating”
• Macrophagy
o Begins when an organelle or other
structure becomes wrapped in a double
membrane from ER
o Resulting vesicle is an autophagic
vacuole or autophagosome
• Macrophage
o Involves formation of smaller
autophagic vacuole surrounded by
single phospholipid bilayer
o Encloses small bits of cytoplasm rather
than whole organelles
Extracellular Digestion
• Discharge on enzymes to the outside of cell by
exocytosis
• Ex. Fertilization of animal eggs
o Head of sperm release lysosomal
enzymes capable of degrading barriers
preventing contact between sperm and
egg
Plant Vacuole
• Acidic membrane-enclosed compartments
o Resemble lysosomes
• Most of the components synthesized in the ER
and transferred to Golgi for further processing
• Provacuole
o Analogous to endosome
o Where coated vesicles convey lipids and
proteins destined for the vacuole
o Matures to a vacuole
• Confines hydrolitic enzymes
• Accumulate solutes, causing water. To enter the
cell by osmosis
o Helps maintain turgor pressure
▪ Prevents plant cells from
collapsing
▪ Drives remodeling of plant cell
• Softening of cell wall—accompanied by higher
turgor pressure— allow plant cells to expand
• Regulates cystolic pH
o ATP-dependent proton pumps
compensate for decline in cytosolic pH
by transferring protons from the cytosol
to lumen of vacuole
• Serves as storage for proteins in seeds
o Specialized type of vacuole
• Other substances in vacuole
o Malate in CAM plants
o Antocynanins (coloring)
o Toxic substances (protection)
o Ionorganic and organic nutrients (shield
from UV)
o Residual indigestible waste
• Stores soluble and insoluble waste
o Plants without mechanism to excrete
soluble waste
Peroxisomes
• Also discovered by Christian de Duve and
colleagues
• Involved in hydrogen peroxide metabolism
• Also bound be single membranes
• Not part of the endomembrane system as they
are not derived from the ER
• Found in all eukaryotic cells
o Prominent in mammalian kidney and
liver cells
o Algae and photosynthetic cells of plants
o Germinating seedlings
• Defining characteristic is the presence of
catalase
o Enzymes esentional for degradation of
hydrogen peroxide (H2O2)
o Potentially toxic compound produced by
oxidative reactions catalyzed by
oxidases
• Both catalase and oxidase are confined in the
peroxisomes
 o Generation and degradation of H2O2
occur within it
• Crystalline core
o Crystalline form of urate oxidase in
peroxisomes
o May consist of catalase in plants
• Diaminobenzidin (DAB) reaction
o Cytochemical test for catalase
Function of Peroxisomes
• Hydrogen peroxde metabolism
• Detoxification of harmful compounds
• Oxidation of fatty acids
• Metabolism of nitrogen-containing compounds
• Cataboism of unusual substance
Hydrogen Peroxide Metabilism
• Oxidases that generate H2O2 transfer electrons
+ hydrogen atoms from their substrates to
molecular oxygen (O2) reducing it to H2O2
o RH2= oxidazable substrate
• Catalase detoxfies two molecules of H2O2
simulataneously either by:
o Oxidizing to oxygen
o Reducing to water
• Catalase can also function as peroxidase where
electrons derived from organic donor are used to
reduce H2O2 to water
• Result is the same in either case: H2O2 is
degraded without leaving the peroxisomes
Detoxification of Harmful Compounds
• As a peroxidase, catalase can use a variety of
toxic substances as electron donors
o Methanol, ethanol, formic acid,
formaldehyde, nitrites, phenols
• All these compounds are harmful to the cell
o Their oxidative detoxification by catalase
is a vital peroxisome function
• Also important in detoxification of reactive
oxygen species
o H2O2, superoxide anion (O2–), hydroxyl
radical (OH•, dot unpaired highly
reactive electron), and organic peroxide
conjugated
o Formed in the presence of molecular
oxygen during normal cell metabolism
o Accumulation lead to oxidative stress
• Perixosomal enzyme for detoxification of
reactive oxygen species
o Superoxide dismutase
o Catalase
o Peroxidase
Oxidation of Fatty Acids
• Via  oxidation
• Provide energy for the cell
• In animal cells, used to degrade long-chain (16-
22 C), very long chain (24-26 C) and branched
fatty acids
• Primary product is acetyl-CoA
o Exported to cytosol where it enters citric
acid cycle
Metabolism of Nitrogen-Containing Compounds
• Most animals (except for primates) require urate
oxidase (uricase) to oxidize urate
o Purine formed during catabolism of
nucleic acids and some proteins
• Urate oxidase catalyzes direct transfer of
hydrogen atoms from substrate to molecular O2
generating H2O2
• H2O2 is immediately degraded in the
peroxisome by catalase
• Allantoin further metabolized and excreted as
allantoic acid or urea
• Aminotransferases
o Catalyze the transfer of amino groups
(—NH3+) from amino acids to -keto
acids
Catabolism of Unusual Substances
• Rare compounds which cell has no other
degradative pathways
o D-amino acids
▪ Not recognized by enzymes
degrading L-amino acids
• Xenobiotics
o Enzymes that break down unusual
substances
Plant Cell Peroxisomes
Leaf Peroxisomes
• In close contract with chloroplasts and
mitochondria
• Involved in glycolate/photorespiratory pathway
• Peroxide-generating oxidase and 2
aminotransferase are confine here
o Enzymes in mentioned pathway
Glyxysomes
• Occurs transiently in seedlings
• Store carbons and energy reserves in seeds as
fat (primarily triacylglycerols)
• Stored triacylglycerols are mobilized and
converted to sucrose during early
postgerminative development
o Includes  oxidation of fatty acids and
glyoxylate cycle
• Found only in tissues where fat is stored
o Endosomes or cotyledons
Peroxisome Biogenesis
• Biogenesis
o Proliferation of organelles
• Peroxins
o Peroxisomal proteins required for
biogenesis
• Previously thought to occur from vesicles similar
to endosomes and lysosomes
• Later, believed to occur from division of
preexisting peroxisomes
• Recent evidence suggest either method or
mixture of both
• Lipids come from:
o Synthesis by peroxisomal enzymes
o Synthesis from ER
• Proteins destined for peroxisome synthesized on
free ribosomes and incorporated after translation
via posttranslational import
Cotranslational Import
• (2) Polypeptides for peroxisomal membrane
enzymes are synthesized on cystolic ribosomes
and threaded through membrane via
transmembrane peroxin transport protein
• (3) Heme enters via a separate pathway,
catalase polypeptides are folded and assemblies
with it
o Form active tertrameric protein
Protein Targeting and Sorting
• Compartments of eukaryotic cells
o Endomembrane system
o Cytosol
o Mitochondria, chloroplasts, peroxisomes
and interior of nucleus
• Polypeptides encoded by nuclear genes routed
to these compartments via diff. mechanisms
• Cotranslational import
o Transfer of polypeptides into ER
o Movement of polypeptide across or into
ER is directly coupled with translation
process
• Postranslational import
o Uptake by organelles of completed
polypeptides requiring the presence of
special targeting signals
• Cotranslational import into ER is the first step in
pathway for delivering newly synthesized protein
to various locations in endomembrane system
• Proteins are synthesized on ribosomes that
become attached to the ER shortly after
translation begins
• Role of ER
o Suggested by Colvin Redman and David
Sabatini
o Newly forming polypeptides pass into
the lumen of ER as they are being
synthsized
▪ Allows them to be routed
through the ER to their correct
destination
• Signal hypothesis
o Model by Günter Blobel and David
Sabatini (1971)
o For polypeptides destined for the ER,
the first segment of the polypeptide to
be synthesized, the N-terminus,
contains an ER signal sequence
• ER signal sequence
o Directs the ribosome-mRNA-polypeptide
complex to the surface of the rough ER
• Complex anchors on the rough ER as a protein
“dock” on the its surface
• Polypeptide chain elongates during mRNA
translation
• It progressively crosses the ER membrane and
enters the ER lumen
• César Milstein and associates
o Evidence for existence of ER signal
sequences
o Studies synthesis of small subunit (light
chain) of immunoglobulin G
o mRNA encoding the immunoglobulin
light chain directs the synthesis of a
polypeptide product that is 20 amino
acids longer at its N-terminal end than
the authentic light chain itself
o Adding ER membranes (microsomes)
lead to the production of an
immunoglobulin light chain of the correct
size
• Findings suggest that extra 20-amino acid
segment is functioning as an ER signal
sequence
o Removed when polypeptide moves into
the ER
• Preproteins
o Proteins containing such ER signal
sequences at N-terminus
• ER signal sequences typically 15-3 amino acids
long and consists of three domains:
o Positively charged N-terminal region
▪ May promote interaction with
hydrophilic exterior of ER
membrane
o Central hydrophobic region
▪ Facilitate interactio of signal
sequence to membrane’s lipid
interior
o Polar region adjoining site where
cleavage from mature protein occurs
Signal Recognition Particle (SRP)
• Mediates contact between ER signal sequence
and ER
• Recognizes and binds to ER signal sequence of
newly forming polypeptide then binds to ER
membrane
• Consists of 6 polypeptides complexed with a
300-nucleotide (7S) molecule of RNA
• Active sides of protein components
o One recognizes and binds to ER signal
sequences
o One that interacts with ribosome to
block further translation
o One that binds to ER membrane
• Process begins when an mRNA endocoding for
a polypeptide destined for the ER starts to be
translated
• (2) Translocons
o Special structure in ER membrane
o SRP binds to it to carry out translocation
of polypeptides across the ER
membrane
o SRP receptor
▪ Where SRP binds
o Ribosome receptor
▪ Holds ribosome in place
o Pore protein
▪ Forms a channel through which
the growing polypeptide can
enter ER lumen
▪ Formed by 3 subunits forming
Sec61 complex
o Signal peptidase
▪ Enzyme that removes ER signal
sequence
 • (3) Sec61 has a central channel that opens as
signal sequence is inserted
Protein Folding and Quality Control in ER
• Molecular chaperones facilitate folding and
assembly of polypeptides
o Occurs after release in ER lumen
• BiP (binding protein)
o Part of Hsp70 family
o Most abundant chaperone in ER
lumen
o Acts by binding to hydrophobic
regions of polypeptide chain
▪ Especially in regions of
tryptophan, phenylalanine,
and leucine
o Prevents aggregation by transiently
binding to hydrophobic regions of
unfolded polypeptide
▪ Stabilizes them
▪ Prevents interaction with
other unfolded polypeptides
• BiP releases polypeptide chain accompanied by
ATP hydrolysis
o Allows polypeptide brief opportunity to
fold (possibly aided by other
chaperones)
• If hydrophobic segments failed to fold properly
BiP binds again and cycle is repeated
• Protein disulfide isomerase
o Facilitates formation of disufide bonds
between cysteines during folding
o Enzyme present in the ER lumen
o Catalyzes formation and breakage of
cysteine bonds
o Acts before synthesis of newly forming
polypeptide is completed
• Unfolded protein respons (UPR)
o Uses sensor molecules in ER
membrane to detect misfolded protein
o Activate signaling pathways that shut
down synthesis of most proteins
o Enhances production of requirements
for protein folding and degredation
• ER-associated degredation (ERAD)
o Recognizes misfolded or unassembled
proteins
o Exports or “retrotranslocates” them back
to the ER membrane to the cytosol
where they are degraded by
proteasomes
Proteins Released into ER Lumen Routed to Golgi,
Secretory Vesicles, Lysosomes, or Back to ER
• Glycoproteins
o Proteins with covalently bound
carbohydrate groups
o Most of the proteins synthesized by
ribosomes on ER
• In ER— glycosylation and folding
• Golgi— further glycosylation and processing of
carbohydrate side chains; site for sorting and
distributing proteins to other locations
• For soluble proteins, default pathway: Golgi—>
secretory vesicle—> cell surface—> fusion w/
plasma membrane
• Proteins entering Golgi not destined for
secretion possess specific carbohydrate side
chains and/or short amino acid signal sequneces
o Target each protein to its appropriate
location within endomembrane system
• Signaling mechanism if proteins final destination
is ER
o C-terminus chanting KDEL sequence
(Lys-Asp-Glu-Leu) or closely related
sequence
o Golgi employs receptor protein that
binds to KDEL and delivers targeted
protein back to ER
▪ Example of proteins: protein
disulfide isomerase and BiP
Anchoring of forming peptide chains to lipid bilayer of
ER membrane
• Done via transmembrane segments of proteins
• Main mechanisms
o Stop-transfer sequences
o Internal start-transfer sequences
Stop-Transfer Sequences
• Invovles polypeptides with typical ER signal
sequence at N-terminus
o Allow SRP to bind to ribosome-mRNA
complex to ER membrane
• Elongation of poly peptide chain continues until
hydrophobic transmembrane segment is
synthesized
• Stretch of amino acids functions as stop-transfer
sequence
o Halts the translocation of polypeptide
through the ER membrane
• Translation continues but the rest of polypeptide
chain remains on cytosolic side of ER
o Result in transmembrane protein
characterized by:
▪ N-terminus in the ER lumen
▪ C-terminus in the cytosol
 • Hydrophobic stop-signal moves latterally out
through the side opening in the translocon and
into lipid bilayer
o Forms the permanent transmembrane
segment anchoring protein to membrane
▪ Type I transmembrane proteins
Internal Start-Transfer Sequences
• Involves membrane proteins that lack a typical
signal sequence at N-terminus
• Instead posses an internal start-transfer
sequences
transfer sequence is traded into the
translocon
o (3) Threading continues untol the next
stop-transfer sequence is encoutered
▪ Next transmembrane section is
released laterally into ER
membrane
o Process continues until all membrane-
spanning sections are inserted
• Incorporated polypeptide can either:
o Remain as an ER membrane protein
o Be transported to other components of
the endomembrane system

Postranslational Import

• Acts as an ER signal sequence that allows and

SRP to bind the ribosome-mRNA complex to
ER membrane
• Hydrophobic region functions as a membrane
anchor that moves through a side opening in
the translocon and permanently embeds
polypeptide in lipid bilayer
• Results in proteins whose C-terminus is in the
ER lumen and N-terminus in the cytosol
o Type II transmembrane proteins
• Threading of multipass proteins into membrane:

o (1) Stop-transfer sequences halts

insertion and releases the first section
of membrane-spanning amino acids
laterally to the ER membrane
o (2) additional start- and stop- transfer
sequences come into play as next start-
 • Uses the same components as contranslational
imports with other differing components
Postranslational Import to Enter Mitochondria and
Chloroplasts
Importing Polypeptides into Mirochondria and
Chloroplasts
• Most polypeptides are synthesized on cytosolic
ribosomes, released into the cytosol, and taken
up by appropriate organelle
• Transit sequence
o Targeting signal for polypeptides
entering mitochondria or chloroplast
o Located at the N-terminus of the
polypeptide
o Contains hydrophobic and hydrophilic
amino acids
• Transit peptidase
o Removes transit sequence once
polypeptide is inside the mitochondrion
or chloroplast
• After transit sequence binds to its receptor,
polypeptide is translocated across outer
membrane through a pore in TOM/TOC
• If destined to interior of organelle quickly
followed by passage to TIM/TIC complex
• Contact site
o Where polypeptide passes between
outer and inner membranes
• Polypeptides interian mitochondria/chloroplast
generall in an unfolded state
• To maintain unfolded state, binding to
chaperone proteins required
• Model for chaperone mediated import of
polypeptides in mitochondria:

• TOM
o Translocase of the outer mitochondrial
membrane
• TIM
o Translocase of inner mitochondrial
membrane
• TOC
o Translocase of the outer chloroplast
membrane
• TIC
o Translocase of the inner chloroplast
membrane
• Transit sequence receptors
o Polypeptides initially selected for • (1) Chaperones of Hsp70 class bind to newl
transport into mitochondria or forming polypeptide being synthesized in
chloroplasts by components of TOM or cyotosol
TOC o Kept in a loosely folded state
 • (2) Transit sequence at N-terminus binds to
receptor of TOM
o Protruding from surface of outer
mitochondrial membrane
• (3) Chaperone proteins released accompanied
by ATP hydrolysis
o Polypeptide is translocated through
TOM and TIM pores into mitochondrial
matrix
• (4) Transit sequence emerges into matrix and
removed by transit peptidase
• (5) mitochondrial Hsp70 molecules binds to
polypeptide entering matrix temporarily
o ATP hydrolysis required for subsequent
release Hsp70
• (6) Mitochondrial Hsp60 chaperone molecules
bind to polypeptide and help achieve its fully
folded conformation
• Mitochondrial import is driven by:
o ATP hydrolysis
o Electrochemical gradient across inner
membrane
▪ Necessary only for binding and
penetration of transit sequence
• Chloroplasts maintain electrochemical gradient
across the thylakoid membrane but not the inner
membrnae
o Energy requirement for import into
chloroplas is met by ATP alone

Targeting Polypeptides to Proper Compartments Within

Mitochondria and Chloroplasts
• Mitochondrial compartments
o Outer membrane
o Inter membrane space
o Inner membrane
o Matrix
• Chloroplast compartments
o Outer membrane
o Inter membrane space
o Inner membrane
o Storma
o Thylakoid membrane
o Thylakoid lumen
• Peptide needs to pass to multiple compartments
to reach final destination
• Given complexity of organelles, polypeptides
require more that’s one signal to arrive at proper
destination