Chapter 12:

The Endomembrane System

Endomembrane System

• Found in eukaryotes only

• Peroxisomes – responsible for hydrogen peroxide-generating reactions and diverse
metabolic functions
• Rough ER – synthesizes proteins
• Smooth ER – synthesizes lipids
• Endomembrane system is composed of (Peroxisomes are not included)
o Endoplasmic reticulum (ER)
o Golgi apparatus
o Endosomes
o Lysosomes
o Associated with both, plasma membrane and nuclear membrane

12.1 The Endoplasmic Reticulum

Endoplasmic reticulum

• Continuous network of flattened sacs, tubules, and associated vesicles throughout the
cytoplasm of the eukaryotic cell
• “Endoplasmic” = within the (cyto)plasm
• “Reticulum” = network
 • Two important parts found in the ER
o ER cisternae (singular: ER cisterna)
▪ Membrane bounded sacs
o ER lumen
▪ Spaces that enclose cisternae
▪ 50-90% of the cell surrounds the ER lumen
• Not visible in the light microscope until stained with dye or fluorescent and improved
resolving power allowed the further understanding of ER
• Functions
o Biosynthesis of lipids, includes triacylglycerols, cholesterol, and related
compounds
o Sources of most lipids that are assembled to form intracellular membranes and
the plasma membrane

Note: Enzymes in the ER are responsible for biosynthesis of proteins for the cell and for export
but not the ER itself
Recall: cytoplasm = cytosol + all other components; cytosol = liquid portion of the cell

The Two Basic Kinds of Endoplasmic Reticulum Differ in Structure and Function

• The two kinds of ER are distinguished by the presence of absence of attached

ribosomes
• Rough endoplasmic reticulum (rough ER)
o Contains large flattened sheets
o Closer to the nucleus compared to smooth ER
o Ribosomes are attached on the cytosolic side of membrane (side that faces away
from the ER lumen) – cisternae side is found hugging and continuous with the
nuclear membrane
o Ribosomes contain RNA which react strongly with basic dyes which makes it
easier to identify the rough ER
o Transitional elements (TEs)
▪ Subdomain of the rough ER
▪ Forms transition vesicles which shuttle lipids and proteins from the ER to
the Golgi apparatus
▪ Often resemble the smooth ER
• Smooth Endoplasmic Reticulum (smooth ER)
o Contains tubular structures
o Absence of ribosomes attached to the membrane and has other roles
• The two are seen to be interconnected which allows material transport without vesicles
• Cells involved in the biosynthesis of secretory proteins and digestive enzymes like the
liver tend to have rough ER networks
• Cells producing steroid hormones such as testis or ovary, contain extensive networks
of smooth ER
Rough ER is Involved in the Biosynthesis and Processing of Proteins

• Ribosomes attached to the rough ER synthesize membrane-bound and soluble proteins

for the endomembrane system
• How do proteins enter the ER lumen from their site of synthesis (cytosolic side of the
rough ER)
o Cytosolic ribosomes synthesize proteins which attach to the rough ER via
receptor proteins in the ER membrane after translation initiation
o The synthesized proteins enter co-translationally (translocation of growing protein
to a certain part of the cell)
▪ Inserted through a pore complex in the ER membrane into the rough ER
lumen as the polypeptide is synthesized by ER-bound ribosome (for
binding)
o After biosynthesis of proteins, membrane-spanning proteins remain anchored to
the ER membrane either by hydrophobic regions of the polypeptide or by
covalent attachment to membrane lipids
o Soluble proteins, including secretory proteins are released into the ER lumen
• The rough ER is the site for several other processes
o Glycoprotein formation
o Folding of polypeptides
o Recognition and removal of misfolded proteins
o Assembly of protein complexes
o ER specific proteins include enzymes that catalyze cotranslational and
posttranslational modifications like glycosylation which is important for sorting
proteins to their proper destinations – essential for proper protein folding
o Quality control
▪ ER-associated degradation (ERAD) – improperly modified, folded, or
assembled are exported from the ER for degradation by cytosolic
proteasomes instead of moving to the Golgi apparatus
▪ Inhouse = stationary genes in the organelle that are synthesize in the
organelle but can be modified and comes back
▪ Examples
• Familial hypercholesterolemia are associated with defects in these
processes

Smooth ER is Involved in Various Processes

• Drug detoxification
o Involves enzyme-catalyzed hydroxylation (addition of hydroxyl groups) makes
hydrophobic drugs more soluble and easier to excrete from the body
o Hydroxylation of organic acceptor molecules in typically catalyzed by a member
of the cytochrome P-450 family of proteins
▪ These proteins are prevalent in the smooth ER of hepatocytes (liver cells)
in which drugs are detoxified
o Medyo tinamad ako magadd ng biochem part dito
 o Pharmacogenetics
▪ Investigates how inherited differences in genes (and their resulting protein
products) can to differential responses to drugs and medications
• Carbohydrate Metabolism – presence of Glucose-6-phosphatase
o Enzymatic breakdown of stored glycogen by the presence of glucose-6-
phosphatase (a membrane-bound enzyme unique to the ER)
o Subcellular fractionation is used to identify ER or visualize fluorescent antibodies
o Glucose-6-phosphate hydrolyzes the phosphate group from glucose-6-phosphate
to form free glucose and inorganic phosphate

o Liver stores glucose as glycogen in the smooth ER and when glucose is needed
by the body, glycogen breaks down by phosphorolysis, producing glucose-1-
phosphate
o This is then converted to glucose-6-phosphate by phosphoglucomutase
o Glucose-6-phopshate must be converted to free glucose by glucose-6-
phosphatase
o Free glucose leaves the liver cell via GLUT2 transporter (recall) and moves into
the blood for transport to other cells

o Glucose-6-phosphatase is present in liver, kidney, and intestinal cells

o Muscle and brain cells retain glucose-6-phosphate to meet their energy needs
• Calcium Storage
o Sarcoplasmic reticulum in muscle cells/ myocytes (along with osteocytes) is an
example of smooth ER that specializes in storing calcium
o In these cells, ER lumen contains high concentrations of calcium-binding proteins
 o Calcium ions are pumped into the ER by ATP-dependent calcium ATPases and
are released in response to extracellular signals to aid in muscle contraction
o Binding of neurotransmitter molecules on the surface of a muscle cell triggers a
signaling cascade leading to release of calcium from sarcoplasmic reticulum –
causing the contraction of muscle fibers
o Calcium is needed for endocytosis, transport, secretory vesicle formation, etc.
• Steroid Biosynthesis
o Smooth ER in certain cells is the site of biosynthesis of cholesterol and steroid
hormones such as cortisol, testosterone, and estrogen
o Examples
▪ Cortisol-producing cells of the adrenal glands
▪ Leydig cells of the testes producing testosterone
▪ Cholesterol-producing cells of the liver
▪ Follicular cells of the ovary producing estrogen
▪ Plastids like elaioplasts
• plants have phytohormones which are synthesized in smooth ER
o Hydroxymethylglutary-CoA reductase (HMG-CoA reductase)
▪ Committed step in cholesterol biosynthesis
▪ Present in large amounts in the smooth ER of liver cells
▪ Enzyme is targeted for inhibition by cholesterol-lowering drugs known as
statins
▪ Smooth ER contains P-450 monooxygenases that is involved in the
synthesis of cholesterol but also in its conversion into steroid hormones
by hydroxylation

The ER Plays a Central Role in the Biosynthesis of Membranes

• ER is the primary source of membrane lipids, including phospholipids and cholesterol

• Some exemptions in biosynthesis of phospholipids other than in the ER
o Mitochondria – phosphatidylethanolamine by decarboxylating imported
phosphatidylserine
o Peroxisomes – have enzymes to synthesize cholesterol
o Chloroplasts – contain enzymes for the synthesis of chloroplast-specific lipids
• Biosynthesis of fatty acids for membrane phospholipid molecules occurs in the
cytoplasm
o Incorporation is restricted to the monolayer of the ER facing the cytosol
• Since phospholipids are synthesized outside the ER, phospholipid translocators or
flippases, catalyze the translocation of phospholipids through ER membrane
o Specific and affect only the rate of a process
o Contribute to membrane asymmetry
• Phospholipid exchange proteins (phospholipid transfer proteins)
o Convey phospholipid molecules from the ER membrane to the outer
mitochondrial and chloroplast membranes
o Transfer of proteins also contribute to the movement of phospholipids from the
ER to other cellular membranes, including the plasma membrane
12.1 The Golgi Apparatus
Golgi apparatus or Golgi complex

• Camillo Golgi, Italian biologist, in 1898

• Functionally and physically linked to the ER (vesicular connections)
• Series of flattened membrane bounded cisternae (disk-shaped sacs stacked together)
o Golgi stack – a series of cisternae (3-8) that varies with the cell type and
metabolic activity of the cell
o Active secretory cells contain hundreds or thousands of Golgi stacks
• ER and Golgi are surrounded by numerous vesicles that carry lipids and proteins from
the ER to various locations like
o Golgi apparatus
o between the cisternae of a Golgi stack
o Golgi apparatus to various destinations in the cell including lysosomes, and
secretory granules
• Intracisternal space is part of the endomembrane system’s network of internal spaces

The Two Faces of the Golgi Stack

• Cis face is oriented towards the ER

• Golgi compartment closest to the ER is a network of flattened, membrane-bounded
tubules referred as cis-Golgi network (CGN)
o Vesicles containing newly synthesized lipids and proteins from the ER arrive at
the CGN, where they fuse with CGN membranes
• Opposite side of the Golgi apparatus is trans-Golgi network (TGN)
o Similar morphology with CGN
o Proteins and lipids leave the Golgi in transport vesicles that bud from the tips of
the TGN cisternae
▪ Transport vesicles carry lipid sand proteins from Golgi apparatus to
secretory granules, lysosomes, and plasma membrane
• Medial cisternae of the Golgi stock
o central sacs between the CGN and TGN comprise of this
 o Processing of protein occurs here
• The CGN, TGN, and medial cisternae are biochemically and functionally distinct
o Each compartment contains specific receptor proteins and enzymes necessary
for specific steps in protein and membrane processing
• Grasp65 genes may be present in Golgi stacks
o Key role in sorting and modification of proteins exported from the ER
o Establishes the stacked structure of the Golgi apparatus or holds them together

Secretory Vesicles

• Going to the plasma membrane and it will be exocytosed it is called a secretory vesicle
• If it will transport intracellularly, they are called transport vesicles
• Different ways of naming transport vesicles
o Rough ER to Golgi apparatus – transitional vesicle
o Shuttle vesicles

Two Models Depict the Flow of Lipids and Proteins Through the Golgi Complex

• Vesicular Cisternae Model

o Each cisterna is a stable structure
o Mediated by shuttle vesicles that buds of from the cisterna and fuses with the
next cisterna in the cis-trans sequence – transports protein packages from one
cisterna to the next
o No changes in the cisternal membrane of the Golgi
o Proteins destined for the TGN are simply carried forward by shuttle vesicles while
molecules in the ER and successive Golgi compartments are retained
• Cisternal Maturation Model
o Transient compartments that change from TGN or CGN
o There are actual changes in the Golgi stack from CGN to TGN
o Transition vesicles from the ER converge to form the CGN, which accumulates
specific enzymes for the early steps of protein processing
o Cis cisterna -> intermediate medial cisterna -> trans cisterna as it acquires more
enzymes
o The TGN forms transport vesicles or secretory granules containing sorted cargo
targeted for various destinations beyond the Golgi
• The diagrams for the model are as follows:
Anterograde and Retrograde Transport

• Anterograde (“front step”)

o Flow of vesicles ER to Golgi
o Discharges through exocytosis, a bit of membrane that originated in the ER
becomes a part of the plasma membrane
• Retrograde (“back step”)
o Flow of vesicles from Golgi cisternae back toward the ER
o To balance the flow of lipids toward the plasma membrane and to ensure a
supply of components for forming new vesicles, the cell recycles lipid sand
proteins no longer needed during the late stages of anterograde transport

12.3 Roles of the ER and Golgi in Protein Glycosylation

• Importance of glyco part – serves as molecular tag

• Glycosylation – addition of carbohydrate side chains to specific amino acid residues,
forming glycoproteins
• Two general kinds
o N-linked – addition of oligosaccharide side chain to the nitrogen on terminal
group of asparagine residues
o O-linked – addition of oligosaccharide side chain to the oxygen on the hydroxyl
group of serine or threonine residues
• The presence of a defective enzyme can block further modification leading to disease
• Ribosomes and synthesized materials are translocated by flippases to be moved
towards the lumen

Initial Glycosylation Occurs in the ER

 • Biosynthesis of core oligosaccharide for N-linked glycosylation of certain asparagine
residues
• Initial processing of core oligosaccharide
• Identification and removal of misfolded proteins
Note: When a glycoprotein becomes 3D, there are more receptors or ligands which serve as
attachment for other molecules making it capable of more biological functionality

Glycosylation in the ER (refer to image next page)

• N-glycosylation will be focused

• The steps of glycosylation may occur as a glycoprotein travels from the ER to the CGN
and through the Golgi apparatus to the TGN
• Occurs in the ER lumen
• Prior to addition of carbohydrate side chains added to proteins have a common core
oligosaccharide
o This consists of 2 units of N-acetylglucosamine (GlcNAc), nine mannose units,
and three glucose units
• Process of glycosylation
o Dolicholphosphate, an oligosaccharide carrier, is inserted into the ER membrane
o GlcNAc and mannose groups are then added to the phosphate group of dolichol
phosphate
o The growing core oligosaccharide is translocated from the cytosol to ER lumen
by flippase
o Once inside the ER lumen, mannose and glucose units are added
o The completed core oligosaccharide is transferred as a single unit from dolichol
to an asparagine residue of the recipient protein
o Core oligosaccharide attaches to a protein is trimmed and modified
▪ Usually core oligosaccharide gets added to a protein while polypeptide is
being synthesized by a ribosome bound to the ER membrane
• Cotranslational glycosylation
o Helps promote proper protein folding
• The inhibition of glycosylation leads to misfolded, aggregated proteins
• Addition of a single glucose unit allows other ER proteins to interact with the newly
synthesized glycoprotein to ensure its proper folding
• One of the two ER proteins Calnexin (membrane-bound) or Calreticulin (soluble) can
bind to monoglucosylated glycoprotein and promote proper folding by forming a complex
with a glycoprotein and thiol oxidoreductase known as ERp57 (catalyzes disulfide bond
formation)
• The protein complex dissociates, final glucose unit is removed by glucosidase II
• A specific glucosyl transferase in the ER known as UGGT (UDP-glucose: glycoprotein
glucotransferase) acts as a sensor for proper folding of newly synthesized glycoproteins
o Binds improperly folded proteins and adds a single glucose unit which makes a
substrate for further binding by calnexin/calreticulin and disulfide formation and
once proper conformation is achieved, new glycoprotein leaves ER to Golgi
Note: Oligosaccharides are continuously added here in ER glycosylation
Proteins Associated in Protein Folding

• Molecular chaperones
o Calnexin and Calreticulin
o Needed for translocation or quality control mechanisms
• Calnexin
o Assist or guides in protein folding and quality control and properly folded and
assembled proteins and N-linked glycosylated proteins travel to the golgi
• Calreticulin
o Binds to the misfolded and misassembled proteins to prevent them from being
exported from the ER to the Golgi apparatus
• Erp57
o Protein trimming
• All are inhouse proteins associated to the ER
• Too big transitional vesicles = use up too much membrane

BiP Molecular Chaperone

• BiP binds to proteins and maintains them in a state of competency

• Essential component for translocation machinery
• BiP translocates molecules from the ER lumen to the ER cisternae (opposite of flippase
since flippase moves something from outside to inside)
Further Glycosylation Occurs in the Golgi Complex

• Correctly trimmed and well-regulated glycosylation products will move to the Golgi
complex
• Reaching the Golgi apparatus
o Connects all the necessary components in Golgi complex and by the time it is
needed to be transported to its specific location, it is a complete package
eventually the organelle has to make it work instead rather than assembling it
alone
• In the CGN
o Attachment of glycosylated proteins
o 1st step: phosphorylation of lysosomal protein (example used in the model)
o Removal mannose which came from the ER (completed its activity of tagging)
▪ Mannose in glycoprotein will be a tag that it’s address of delivery is going
to the Golgi and Golgi only
▪ Different oligosaccharide – different location
▪ Tags can be protein (lysosome), carbohydrate, or lipid (+ protein to the
plasma membrane)
• In the TGN
o Addition of sialic acid (lysosome contains acid hydrolases which requires the
addition of acids)
o Terminal glycosylation occurs here

Terminal Glycosylation

• Allows more diversity in structure and function of oligosaccharide side chains

 • Removal of a few carbohydrate units of the core oligosaccharide or addition of GLcNAc
and other monosaccharides like galactose or sialic acid
• Targets of bacterial and viral components which leads to complications
• Glycosyl transferases
o Addition of galactose units
o A marker enzyme unique to the Golgi
• Two important enzymes in Golgi stacks
o Glucan synthases – produce oligosaccharides from monosaccharides
o Glycosyl transferases – attach carbohydrate groups to proteins
▪ ER contains hundred of glycosyl transferases, indicating the potential
complexity of oligosaccharide chains
• Each cisterna contains a distinct set of processing enzymes

12.3 Roles of ER and Golgi Complex in Protein Trafficking

• Each protein contains a specific “tag” targeting the protein to a transport vesicle that will
carry material from one cellular location to another
o The tag may be a oligosaccharide side chain, hydrophobic domain, short amino
acid sequence, or some structure feature
o Tags may be involved in excluding materials from certain vesicles
• Membrane lipids can also be tagged by a phosphate group attached to positions 3,4,
and/or 5 of a membrane phosphatidylinositol (PI) molecule by a specific kinase
o In mammalian cells, inositol kinases disrupt vesicle trafficking to the lysosome
o Length and degree of saturation of membrane lipids have shown to be important
in vesicle trafficking
• General idea or process of Protein Trafficking
o Proteins are tagged -> Proteins are sorted -> Vesicles bud ->Proteins interact
with receptors -> Delivery away from Golgi
• Golgi can also be involved in the processing of proteins that enter the cell by
endocytosis
ER-Specific Proteins Contain Retention and Retrieval Tags

• Protein composition is maintained both by preventing some proteins from escaping when
vesicles bud from the ER membrane and by retrieving other proteins that left the ER and
reached the CGN
• Several proteins localized to the ER contain tripeptide sequence RXR (arg-X-arg; X =
any amino acid) which promote retention in the ER
o This retention tag is found in some multisubunit proteins that are destined for the
plasma membrane
o N-methyl-D-aspartate (NMDA) receptor
▪ Important in neurotransmission in a mammalian brain contains such a tag
o Proper assembly masks the RXR sequence, allowing the assembled complex to
leave the ER
o Phosphorylation of protein near the RXR sequence promotes ER release
• Retrieval tags
o Found in soluble ER-specific proteins
o Bind to specific transmembrane receptors facing the Golgi apparatus and lumen
o KDEL or KKXX in mammals
o HDEL in yeast
o When a protein tag binds to a receptor, it undergoes conformational change, and
the receptor-ligand complex is packaged into a transport vesicle for return to the
ER
• Chimeric proteins
o 2 polypeptide segments from different proteins allowing a hybrid polypeptide to
be produced from the joined DNAs
o When proteins are secreted from the cell were altered in this manner, proteins
were found in the ER rather than being secreted
o This shows that tags do not only prevent escape of the protein but actively
promoted the retrieval of ER specific proteins

• Golgi network is not the only one responsible for trafficking as the cytoskeleton helps as
well as organelle markers can be glycosylated to indicate that a certain protein is needed
elsewhere or should be kept in the organelle by specific tags
 • Sorting occurs in the TGN in the Lumen of the Golgi
• Budding and transport vesicles with respective tags transfer to their specific organelles

Targeting of Soluble Lysosomal Proteins to Endosomes and Lysosomes is a Model for Protein
Sorting in the TGN
 • Lysosomal proteins need to move to lysosomes to be used for intracellular functions
• Lysosomal enzymes like hydrolases are synthesized and mannose-6-phosphate or
mannose will be used as biological tags to ensure delivery towards the lysosomes
• Steps in this process
o In the ER, soluble lysosomal enzymes undergo N-glycosylation followed by
removal of glucose and mannose units
o Within the Golgi apparatus, mannose residues of lysosomal enzymes are
phosphorylated (added a P-) by two Golgi specific enzymes
▪ First, located in early compartments of the Golgi stack,
phosphotransferase adds GlcNAc-1-phosphate to carbon atom 6 of
mannose
▪ Second, in the mild-Golgi compartment, removes GlcNAc, leaving
mannose-6-phosphate residue
o In the TGN, tagged lysosomal enzymes bind to mannose-6-phosphate receptors
(MPRs) in the TGN membrane
▪ pH of TGN = 6.4 pH, favoring binding of soluble lysosomal enzymes to
these receptors
o Receptor-ligand complexes are packaged into transport vesicles and conveyed
to an endosome
▪ In animal cells, lysosomal enzymes needed for degradation of material
brought into the cell by endocytosis and transported from the TGN to
organelles known as late endosomes
▪ These develop from early endosomes which are formed by the
coalescence of vesicles from the TGN and plasma membrane
o In some cells, degradation and recycling of unneeded or damaged cell
components are carried out by specialized late endosomes called multivesicular
endosomes (MVEs) which are sometimes known as multivesicular bodies
(MVBs)
▪ Serve as an intermediate between early endosomes and lysosomes and
contain intraluminal vesicles that form by budding into the interior of
MVEs
▪ MVE can fuse with a lysosome to degrade its contents or material in the
MVE (like membrane-bound receptors proteins) can be recycled
o As early endosome matures to late endosome, pH of the lumen decreases to 5.5,
causing lysosomal enzymes to dissociate from the MPRs
▪ This prevents the retrograde movement of enzymes back to the golgi as
receptors are recycled in vesicles return back to the TGN
▪ Late endosome matures to form a new lysosome or delivers its contents
to an active lysosome
• Human disease named I-cell disease supports this model of lysosomal enzyme targeting

12.5 Exocytosis and Endocytosis: Transporting Material Across the Plasma Membrane

• Two methods transporting materials across the plasma membrane

o Exocytosis – process by which secretory granules release their contents to the
exterior of the cell; final step is secretory pathways
 o Endocytosis – process by which cells internalize external materials
• Unique to eukaryotic cells and involved in the delivery, recycling, and turnover of
membrane proteins

Secretory Pathways Transport Molecules to the Exterior of the Cell

• Key feature of vesicular traffic are secretory pathways by which proteins move from the
ER through the Golgi apparatus to secretory vesicles and secretory granules, which then
discharge to the exterior of the cell
• James Jamieson and George Palade in 1967
• By 37 minutes
o Labeled protein was detected in vesicles budding from the Golgi called
condensing vacuoles
• 117 minutes
o Labeled protein began to accumulate in Zymogen granules (highly concentrated
proteins are found inside this mature regulated secretory vesicle), vesicles that
discharge to the secretory proteins to the exterior of the cell
• Various types of secretion
o Constitutive secretion – secretory vesicles move directly to the cell surface and
fuse with the plasma membrane and release their contents by exocytosis
▪ Unregulated processes, continuous and independent of extracellular
signals
▪ Short amino acid tags identify specific proteins for constitutive secretion
▪ E.g. release of mucus in the intestine
o Regulated secretion – secretory vesicles accumulate in the cell then fuse with the
plasma membrane only in response to extracellular signals
▪ E.g. release of insulin from pancreatic B cells in response to glucose and
release of zymogens (active precursors of hydrolytic enzymes) in
response to calcium or endocrine hormones
o Polarized secretion – secretion of proteins is limited to a specific surface of a cell
 ▪ E.g. intestine releasing digestive enzymes on the side of the cell that
faces the interior of the intestine; nerve cells secrete neurotransmitter
molecules only at junctions with other nerve cells
▪ Proteins, lipids, and protein components of two different membrane layers
are sorted into vesicles that bind to localized recognition sites on
subdomains of the plasma membrane

Exocytosis Releases Intracellular Molecules Outside the Cell

Exocytosis

• Proteins or other materials in a vesicle are released to the exterior of the cell as the
membrane of the vesicle fuses with the plasma membrane
o Animals secrete peptide and protein hormones, mucus, and several digestive
enzymes
o Plant and fungal cells secrete enzymes and structural proteins associated with
the cell wall, and
o Carnivorous plants secrete hydrolytic enzymes to digest trapped insects
• Microtubules are oriented parallel to the direction of vesicle movement and stop when
treated with colchicine, a plant alkaloid preventing microtubule assembly
Role of Calcium in Triggering Exocytosis

• Fusion of regulated secretory vesicles with the plasma membrane is triggered by

extracellular signals like hormones and neurotransmitters which binds to receptors that
triggersthe synthesis or release of a second messenger within the cell
• During regulated secretion, a transient increase of calcium ions that leads the cell to
undergo exocytosis
o Elevation of calcium leads to activation of protein kinases whose target proteins
are components of either the vesicle membrane or the plasma membrane
• E.g. microinjection of calcium to pancreatic cells induces mature secretory granules to
discharge their contents from the extracellular medium

• Lysosomes
o Will phagocytose the bacteria or release the bacteria

Endocytosis Imports Extracellular Molecules by Forming Vesicles from the Plasma Membrane
Endocytosis

• Small segment of the plasma membrane progressively folds inward and then pinches off
to form an endocytic vesicle containing ingested substances of particles
• Important for several cellular processes like ingestion of nutrients by some unicellular
organisms and defense against microorganisms by white blood cells
 • Through endocytosis and retrograde transport, cell can recycle and reuse molecules
deposited in the plasma membrane by secretory vesicles during exocytosis
In terms of membrane flow

• Exocytosis adds lipids and proteins to the plasma membrane

• Endocytosis removes them

• During endocytosis, membrane of endocytic vesicle isolates the internalized substances

from the cytosol
• Most endocytic vesicle develop into early endosomes, which fuse with vesicle from the
TGN, acquiring digestive enzymes and maturing to form new lysosomes
• Phagocytosis = “cellular eating” which large solid particles are ingested
• Pinocytosis = “cellular drinking” in which liquids or suspended molecules are taken up

Phagocytosis

• Ingestion of large particles (> 0.5 um in diameter, including aggregates of

macromolecules, parts of cells, and even whole microorganisms and other cells
• Phagocytosis is a means of acquiring food and nutrients between
o For unicellular prokaryotes like amoebas and ciliated protozoa
o Primitive animals, flatworms, coelenterates, and sponges to obtain nutrients
• In more complex organisms, phagocytosis is restricted to specialized cells called
phagocytes
 • Examples
o Macrophages and neutrophils that use phagocytosis for defense rather than
nutrition as these cells engulf invasive microorganisms in injured tissues or the
blood stream
▪ Macrophages may also act as scavengers, ingesting cellular debris and
whole damaged cells from injured tissues
• Contact with food particles or smaller organisms triggers the onset of phagocytosis

• Pseudopod
o Fold of membrane which gradually surround the object and then meet and engulf
the particle, forming an intracellular phagocytic vacuole
• Phagosome
o The endocytic vesicle called phagosome, fuses with a late endosome or matures
directly to a lysosome forming a large vesicle in which the ingested material is
digested
• As part of their role in immune system, human phagocytes generate toxic concentrations
of hydrogen peroxide, hypochlorous acid, and other oxidants in the phagocytic vacuole
to kill organisms

Receptor-Mediated Endocytosis or Clathrin-dependent endocytosis

• Cells acquire certain soluble and suspended materials by using specific receptors on the
outer surface of the plasma membrane
• Primary mechanism for the special internalize of most macromolecules by eukaryotic
cells
 o Mammalian cells can ingest hormones, growth factors, enzymes, serum proteins,
cholesterol, antibodies, iron, viruses, toxins by this mechanism
• Example
o Hypercholesterolemia – high blood cholesterol levels leading to atherosclerosis
nad heart disease
• Process of receptor-mediated endocytosis

(1) Begins with binding of ligands to their receptors – specific ligand-binding proteins on
the outer surface of the membrane
(2) As receptor-ligand complexes diffuse in the membrane, they encounter specialized
membrane regions called coated pits that serve as sites for collection and internalization
of these complexes

• Coated pits occupy 20% of total surface area of plasma membrane

(3) Accumulation of receptor-ligand complexes within coated pits triggers accumulation
of proteins on the inner (cytosolic) surface of the plasma membrane

• These proteins – including clathrin, clathrin adaptor proteins, and dynamin which
promote membrane curvature and invagination of the pit
 (4) Invagination continues until the pit pinches of the plasma membrane, forming a
coated vesicle
(5) The coat proteins and dynamin are recycled to the plasma membrane and become
available for forming new vesicles while the uncoated vesicle is free to fuse with early
endosomes
(6) Coated vesicles are important in a number of cellular processes that transport
proteins and lipids within the endomembrane system

• First variation of receptor-mediated endocytosis

o Epidermal growth factor (EGC) stimulates division of epithelial cell and
undergoes endocytosis
o EGF receptors are internalized, the cell becomes less responsive to EGF, a
process known as desensitization
▪ Deficiencies in desensitization caused by defective endocytosis can lead
to overstimulation by EGF, resulting in excessive cell growth, cell division,
and possible tumor formation
• Second variation
o Receptors are concentrated in coated pints independent of formation of receptor-
ligand complexes
o Binding of ligands to receptors triggers internalization
• Third variation
o Receptors are not constitutively concentrated, but also constitutively internalized
regardless of whether the ligands have bound to the receptors

Following receptor-mediated endocytosis

• Uncoated vesicles fuse with vesicles budding from TGN to form early endosome in
peripheral regions of the cell
• Early endosomes
o Sites for sorting and recycling of extracellular material brought in by endocytosis
• Proteins are essential for new rounds of endocytosis are often but not always recycled
after separation from the digested material
• Early endosome acquires lysosomal proteins from TGN and matures to form late
endosome
• Increase in pH leads to recycling of plasma membrane receptor molecules in the early
endosome
o Maintained by ATP-dependent proton pump
o Acidic environment decreases the affinity of most receptor-ligand complexes,
thereby freeing receptors to be recycled while newly ingested material is diverted
to other locations (recall mannose-6-phosphate tagging)
• Depending on the ligand, some receptor-ligand complexes do not dissociate in the early
endosome
 o Intact receptor-ligand complexes are subject to sorting and packaging into
transport vesicles
o Three alternatives for these complexes
▪ Some receptor-ligand complexes are carried to a lysosome for
degradation
▪ Others are carried to the TGN, they enter a variety of pathways
transporting material throughout the endomembrane system
▪ Receptor-ligand complex travel by transport vesicles to a different region
of the plasma membrane where they are secreted as part of transcytosis
• This pathway accommodates the transfer of extracellular material
from one side of the cell, where endocytosis occurs, through the
opposite side of the cell, where exocytosis occurs
• Example:
o Immunoglobulins are transported across epithelial cells
from maternal blood to fetal blood by transcytosis

Clathrin-Independent Endocytosis

• An example of clathrin-independent endocytic pathway is fluid-phase endocytosis

o Type of pinocytosis for non-specific internalization of extracellular fluid
o Does not focus on ingested material because the cell engulfs fluid without a
mechanism for collecting molecules or excluding particular molecules, the
concentration of material trapped in vesicles reflects the concentration in the
extracellular environment
• Fluid-phase endocytosis occurs at constant rate in eukaryotic cells
• Because it compensates for the membranes continuously added to the plasma
membrane by exocytosis
• It is a means of controlling a cell’s volume and surface area
• Once inside the cell, fluid-phase endocytic vesicle like this are routed to early
endosomes

12.6 Coated Vesicles in Cellular Transport Processes

Coated vesicles

• Reported by Thomas Roth and Keith Porter in 1964

• Common feature of most cellular processes involving the transfer or exchange of
substances between membrane-bounded compartments or between the inside and
outside of a cell
• Contains a layer or coat of protein on the cytosolic side of the membrane surrounding
the vesicle
• Most studied coat proteins involve clathrin, COPI, COPII (COP = coat protein)
• Involved in vesicular traffic throughout the endomembrane system, as well as transport
during exocytosis and endocytosis
• Roles and functions
 o Helps in sorting of molecules that are fated for different destinations
o Forcing flat membranes to form spherical vesicles
o Prevent premature fusion of budding vesicle with nearby membranes
o Regulating interactions between budding cells and microtubules – for moving
vesicles through the cell
• Clathrin-coated vesicles
o Involved in selective transport of proteins from the TGN to endosomes and in the
endocytosis of receptor-ligand complexes from the plasma membrane
• COPI-coated vesicles
o Facilitate retrograde transport of proteins from the Golgi back to the ER, as well
as cisternae of the Golgi apparatus
• COPII-coated vesicles
o Transport of material from the ER to the Golgi
• Caveolin-coated vesicles
o Cavolae (“little caves”) are small flask-shaped invaginations of plasma
membranes 50 years ago
o Characterized by the presence of cholesterol-binding protein caveolin and
cholesterol uptake by cells
o Mice deficient in caveolin show dramatic abnormalities in the cardiovascular
system which becomes enriched in cholesterol
▪ Target for treatment of cardiovascular disease
o Other roles
▪ Endocytosis and exocytosis, redox sensing and signal transduction,
regulation of airway function to lungs, caveolae contain proteins important
in calcium signal in heart muscle cells

Clathrin-Coated Vesicles Are Surrounded by Latices Composed of Clathrin and Adaptor Protein

• Might wait for sir to further narrow this down