Cell Biology

Dr. Jeneni Thiagavel

Summer 2021
 Lecture 2
Cell
● All living things are made of cells, which are small membrane enclosed units.
● Can make copies of themselves by growing & dividing

Cell Biology: study of structure, function, & behavior of cell

● Diversity of organisms, but on the molecular level the organisms are similar.

Heredity: the passage of information/characteristics from parent to offspring.

● However, cell division could also be heredity, passage of information from one cell to
another (the duplicate of itself).
● Life depends on the cell's ability to store, retrieve, & translate information through
genetic instruction.

Cells store information that is readable by the info handling machinery of cells.
● This information = genetic information
● Cell needs to be able to read this information
● How this information is read is similar across organism
○ Universal genetic code: the fundamental similarity despite the diversity we see on
the morphological level.

Universal Features of Living Cells

1. DNA
○ Deoxyribonucleic Acid (DNA)
○ All cells on earth store hereditary information in
form of DNA
○ Polymer chain
■ Monomer = nucleotides
○ Double helix structure
○ Nucleotide = 5 carbon sugar + phosphate
group + nitrogenous base
○ Sugar = deoxyribose in DNA, ribose in RNA
○ 4 different nitrogenous bases = Adenine,
thymine, guanine, cytosine
■ Sugar & phosphate are attached together = Sugar- Phosphate Backbone
■ The bases themselves are not attached
2. Templated Polymerization
● All cells replicate using template polymerization
❖ Use one strand of DNA to form complementary strand
➢ A pairs with T, C pairs G
➢ Enabled by complementary base pairing between nucleotides.

❖ Hydrolysis
➢ To break the phosphate -phosphatebond
■ EX: from sugar + base + 3 phosphates => sugar + base + 2 phosphates
(GT(tri)P -> GD(di)P
● Nucleotide monophosphate = nucleotide
➢ Key points: very high energy stored in the phosphate -- phosphate bond
■ When you break it off -> energy released -> that energy is used to carry
out different biological processes in cells.
■ Example: energy used to attach nucleotide to the growing complementary
strand in templated polymerization (polymerization reaction)

❖ Nucleoside = sugar + nitrogenous base

➢ 1 phosphate group attached = nucleotide or nucleoside monophosphate
➢ 2 + phosphate groups: nucleoside diphosphate, nucleoside triphosphate

❖ Free Energy
➢ Free energy is important for the cell
■ Life requires free energy
➢ Cell gets energy from food we eat -> mitochondria & oxidative phosphorylation
➢ To grow or cell replication, a cell takes free energy & raw material from the
environment.

3. (and 4) Flow of Genetic Information

❖ The Central Dogma of Molecular
➢ Replication-> Transcription-> Translation
➢ DNA synthesis -> RNA synthesis -> Protein
sysnthesis
■ All cells need to do transcription
■ All cells need to do translation
❖ Cells produce molecules whose chemical properties cause them to self-assemble into the
structure a cell needs.
❖ Amino acids -> chemical properties -> contribute to the overall structure & function of
the protein in the cell.
 5. The Plasma Membrane
❖ All cells enclosed by a membrane
➢ Selective barrier
❖ Amphiphilic,
➢ hydrophobic tails, hydrophilic heads
❖ Not impermeable
➢ Has transport proteins embedded to allow passage of molecules that can’t
be assimilated
➢ Needs to import raw materials and export wastes

❖ Prokaryotes vs Eukaryotes
➢ Eukaryotes
■ keep their genetic information into the nucleus (has the genome & is the
primary site of DNA/RNA synthesis.
■ Bigger (1000 times larger in volume and
10 times bigger in linear dimension) and
more organized than prokaryotic cells
■ Bigger genomes
■ Different in cell structure & function

➢ Prokaryotes
■ Simple & small
■ Spherical or rod-shaped
■ Bacterium: plasma member & cell wall
■ Less organized than Eukaryotes

❖ Major features of Eukaryotic Cells

➢ Nuclear envelope
■ Double membrane to separate nucleus & surrounding organelles
➢ Cytoskeleton
■ Gives cell membrane strength & shape
■ Microtubules, actin filaments, & intermediate filaments
➢ Mitochondrion
■ Enclosed by double membrane
■ Take in food molecules and turn it into form of energy cells can use
■ Similar to bacteria
■ Has its own genome and ribosomes
■ Still depends on the cell
❖ Internal membranes
➢ Nuclear membranes & double membranes around mitochondria
➢ Similar to plasma membrane

❖ Animal cell has plasma membrane but no cell wall

➢ Allows animal cells to change shape and mobility

❖ Chloroplasts
➢ Plant and algae
➢ Similar to mitochondria (double membrane, has its own genome & DNA, still
depends on the cell
➢ Photosynthesis, sun -> energy

❖ Plant cell
➢ Cell wall -> can’t move or engulf other cells, more rigid
➢ Plant cells also have mitochondria, chloroplasts don’t substitute mitochondria
 Lecture 3
More on Eukaryotic cells
❖ They have big genomes
❖ They have more genes than prokaryotes
➢ Transcription & translation at those genes are very specific and controlled
➢ Coding DNA region & non-coding DNA region
■ Coding region = protein, non-coding = will be spliced out in RNA
transcript
● Eukaryotes have more coding regions than prokaryotes

Regulatory DNA
❖ More noncoding DNA than coding DNA
❖ Some noncoding DNA have important functions
➢ Regulatory DNA assembled in the non-coding region
■ Those DNA regulate whether a certain gene will get transcribed
■ Saves energy for the cell

Transcription and Translation

 Controlled
 Depending on which proteins the cell needs, the specific gene that codes that protein will
be “turned on”, and vice versa if there is too much of the protein.

Macromolecules
❖ The cell contains 4 major families of small organic molecules
➢ They are carbon-based compounds
➢ Sugars, fatty acids, amino acids, and nucleotides
■ Monomers
■ building blocks of the cell
❖ Polymeric macromolecules
➢ Polysaccarides, Fats lipids and membranes, Proteins, and Nucleic Acids
➢ Make up large fraction of cell mass
➢ Most abundant carbon-containing molecules in a living cell
➢ Larger units of the cell
❖ Small organic molecules
➢ Less abundant than macromolecules
➢ All organic molecules are synthesized from and can be broken down to the same
simple compounds
 ➢ Others act as energy sources that are broken down
(bonds) and transformed
➢ Can be taken apart of modified and used by the cell
➢ Can be used to construct diverse range of proteins,
nucleic acids, and other macromolecules

Proteins
❖ Polymer chains made of monomers that are the same for all living cells (amino acids)
❖ All organisms use the same 20 types of amino acids
❖ The process of transcription and translation also similar across all organisms

Codon
❖ 3 nucleotides = 1 Codon= 1 amino acid

Protein molecule = one or more polypeptides (many amino acids)

❖ Peptide = 2 or more amino acids
❖ Polypeptide - multiple peptides
❖ Each protein performs its function according to its own genetically specified sequence of
amino acids

#6 All living cells use enzymes as catalysts

Proteins
❖ Perform 1000s of functions in cells
❖ (1) Many serve as enzymes
➢ Enzymes: the biological catalysts (speeds up or allows a reaction to happen) that
facilitate the many covalent bond making and bond-breaking reactions that the
cell needs
➢ Each enzyme catalyzes a particular reaction
■ Ex: DNA polymerase catalyzes the polymerase reaction
➢ Enzyme-catalyzed reaction
■ Chain reaction
 ■ The product of 1 reaction became the substrate (starting material) of
another reaction
■ Interconnected reactions important to form complex organisms

❖ (2) Used to build structural components

➢ Microtubules made from tubulin protein

❖ (3) Act as motor proteins to produce force and movement

➢ Motor proteins walk on microtubules to transport materials inside of cells

❖ (4) Regulate gene expression

➢ Regulatory DNA regions serve as places for transcription activators/repressors
(proteins involved in gene regulation) to assemble
➢ Enhance gene expression or repress it
❖ (5) Cell communication
➢ Ligand and receptor protein
❖ Perform cell functions determined by its AA & 3D structure

Condensation Reaction = bond forming

❖ Adding monomers to the end of a growing
chain or adding monomers together
➢ Lose water in this process
➢ Polymerization reactions (so adding
nucleotide to the growing chain)
➢ *A general term, templated
polymerization = has a template
(like in DNA replication)
❖ Subunits of monomers not identical together (20 different amino acids, 4 nitrogenous
bases)
➢ Not strung together in a random order
➢ The order that the nucleotides are strung together are specified by the RNA
transcript
Hydrolysis = bond breaking + releasing energy
Properties of Macromolecules
❖ Bonds in macromolecules allow rotation of the atoms, so the polymer chain is flexible.
❖ Macromolecules can adopt a variety of conformations , but will have its most
energetically favorable conformation.
❖ Attraction between different macromolecules
➢ Macromolecules = building block to form large structure
➢ Ribosomal RNA + ribosomal protein to make ribosomes.
➢ Different protein molecules can come together to form large assembly too.

Protein Structure
❖ Protein
➢ Cell’s building blocks
➢ Execute most of the cell’s functions

❖ The location of each amino acids that forms a protein determines its 3D shape (structure)

➢ Which influences its fucntion

❖ Polypeptides
➢ Amino acids linked to their neighbors
by peptide bonds (covalent)

❖ Key points
❖ Peptide bonds between the polypeptide
backbone
❖ Amino terminus (N-terminus) & carboxyl
terminus (C-terminus)
❖ Side chain gives an amino acid its unique
properties (hydropho/phi, + or -), which gives the protein its unique properties too.

Gene Expression
❖ DNA -> RNA -> Protein
Protein Folding
❖ Folding of a protein chain determined by diff sets of noncovalent bonds that form
between parts of chain
❖ No 2 atoms must overlap
❖ It is the combined strength of many noncovalent bonds that determine the stability of
each folded shape

❖ Polar covalent bond = can share electrons

❖ Non- polar covalent bond = no dipole moment, no partially positive, no partially negative

❖ Non- Covalent bond

➢ Hydrogen bond = weak attraction between the oxygen of one water molecule &
hydrogen of another molecule
➢ Hydrophobic regions attracted to each other

❖ Hydrophobic Clustering
➢ Governs the folding of any protein
➢ In aqueous environment
■ The nonpolar side chains associate
with each other bc they don’t want to
be in water
■ The polar side chains can interact
with water and form hydrogen bonds
 Lecture 4: Protein Structure Cont.
Protein Structure- Level of Organization
❖ 1.Primary structure- the amino acids connected by peptide bond between the polypeptide
backbone (can’t fold, just the amino acid sequence)
❖ 2.Secondary structure- alpha helix and beta sheet
❖ 3.Tertiary structure- 3D organization, polypeptide
❖ 4.Quaternary- multiple tertiary structure, multiple polypeptides

❖ Most proteins have a 3D structure which is determined by the order of AA in the chain
❖ The conformation is of the lowest energy
➢ It is the shape and structure
❖ Treatments
➢ Solvents can disrupt the noncovalent interactions holding the folded chain
together
➢ Which denatures (lose shape & structure) the protein
➢ Remove that solvent, sometimes protein can fold back to that lowest energy
conformation again (Renature)
❖ Most proteins fold up into a single stable conformation, but this changes slightly when
the protein interacts with other molecules

Molecular Chaperones: a class of protein that most proteins need to fold correctly.
❖ If no chaperones are present:
➢ 1.Proteins may not correctly folded in their most stable form
➢ 2.Non- functional and potentially dangerous structures
■ “Off-pathway conformations”
● Before the proteins fold into its 3D shape, exposed hydrophobic
regions (nonpolar side chains) can bind to exposed nonpolar side
chains on other proteins & irreversibly cluster
● Chaperones prevent this from happening by binding to the exposed
hydrophobic surfaces with their own hydrophobic surfaces
❖ Final 3D shape of a protein = specified by its amino acid sequence (chaperones just make
reaching this folded state more reliable)

Alpha helix and beta sheet

❖ Alpha helix = single polypeptide that fold/twist into themselves, very
common folding patterns
❖ Beta sheets = held together by hydrogen bonds between peptide bonds
in neighbor chains
- Can run in same or opposite (anti- parallel) directions
Coiled- Coil
❖ Multiple alpha helices fold together
❖ Wrap around each other to minimize exposure of hydrophobic amino acid side chains to
aqueous environment
❖ Hydrophobic side chains facing each other, hydrophilic side chains exposed to water .

Why Fold?
❖ Stable conformation favored by natural selection
❖ Unpredictable structures usually eliminated by natural selection
❖ Once folded, its structure can be modified to allow new functions

Protein Families
❖ Proteins that have amino acid sequences and 3D conformations that resemble each other
➢ But have different functions
■ Ex: Elastase and chymotripsin
➢ New variations of proteins are selected usually because of its ability to do
something, neutral function are not gonna be selected again

❖ Paralogs = 2 different proteins belonging to part of the same family in 1 organism

❖ Orthologs = 1 protein in 2 different organisms

Protein Complex: Multiple polypeptide chains that that come together

Protein subunits: Individual polypeptide chains
Protein Domains: structural units that fold independently of each other
❖ Also called interaction domains, sites that interact with other proteins

*In Protein complexes (Multiple polypeptide/Quaternary structure), we called each of those

subunits protein subunits.
*Protein domains are specific regions on the protein complexes that interact with other proteins.
Multidomain Proteins
❖ Domains are usually related structures with related functions
❖ Protein Modules: domains highly conserved throughout evolution
❖ The number of protein domains increases with increased complexity of the organism.
➢ This variety in proteins increases the range of protein-protein interactions.
➢ More interaction domains, so more interactions between other proteins, so more
complex.

Multiple Polypeptide
❖ Each polypeptide chain in this protein is called a protein subunit
❖ Recall: Protein domains can be made up of more than one polypeptide chain, only use the
terms interchangeably if the protein domain is made up of one polypeptide
❖ Binding site: any region of a protein surface that can interact with another molecule (can
be referred to as a domain or protein subunit)
❖

Example: hemoglobin
➔ Is the protein that carries oxygen around in the bloodstream
➔ Hemoglobin has two alpha globulin & two beta globulin proteins that are symmetrically
arranged.

Protein as subunits of Large Structures

❖ Formed by the noncovalent assembly of many separately manufactured proteins
➢ Advantages of having non homogeneous subunits
■ 1.Regulate less genetic information
■ 2.Reversible assembly, can easily disassemble to multiple different pieces
■ 3.Avoid errors
Why do proteins form closed structures?
➢ Advantages of forming closed structures
➢ Additional stability

Protein as subunits of Large Structures

❖ Some proteins can form many shapes such as sheets, like what is found in the lipid
bilayer
❖ Hexagonal packed sheet or tube or sphere shape
❖
Why do proteins form closed structures?
➢ Advantages of forming closed structures
- Additional stability due to the number of bonds between the protein
subunits is increased

Globular Proteins
- Globular Proteins: proteins that fold up into a compact shape
- one can easily make a long chain of identical protein molecules if each subunit can bind
(or has a region that can bind) to another subunit or multiple subunits at once. -
Hemoglobin, immunoglobulin, and protein kinase A are examples of globular proteins.

Helices
- Biological structures are formed by linking similar subunits into long
repetitive chains
- If the subunits are identical, they will fit together in just one way adjusting
their positions to minimize energy loss
- Each subunit is positioned in exactly the same way relative to the next.

Enzymes
- Are usually globular, protein compact, large, complex with many subunits
- Recall Transcription, to make the RNA transcript, it uses RNA polymerase.

Fibrous Proteins
❖ Alpha keratin -> hair & nails
❖ Forms dimer of 2 identical subunits
❖ Globular domains at end of coiled- coil
❖ The domains are binding site that allows the fibrous proteins to bind to other coiled-col to
form intermediate filaments
 ❖ Example of fibrous proteins
➢ Collagen molecule formed by 3 helices
➢ Very tough & connective tissue

Disordered Regions of Proteins

❖ Important functions in the cell’s interior
❖ Loops of polypeptide chain that bind to other molecules
❖ Disordered tails
❖ Protruded out of the core region , unstructured until bind to other molecules
❖ ex: histone octane = 8 subunits & disordered regions, important functions in DNA
packaging

❖ Binding = disordered region can serve as binding site

❖ Signaling = disordered region can be modified
❖ Tethering = disordered region helps protein binds together
❖ Diffusion barrier = disordered region control things passing in & out of nuclear pores
Protein Function
❖ All proteins bind to other molecules
❖ A protein's physical interaction with other molecules determine its biological properties
➢ Tight or weak
➢ High specificity

Protein function 1: Extracellular Signal Molecule

❖ Ligand
➢ Binds to receptor protein
➢ Cell communication
➢ Create signaling cascade in that cell
➢ Ligand + receptor protein binding = weak, non-covalent bond because you don’t
want to have a signaling cascade over and over again
➢ Binding site on the receptor protein very specific
■ Side chains form binding site cavity via noncovalent bonds

More on ligand binding site

❖ Water can compete with the site
❖ Make ligand site dry, so water molecule won’t form hydrogen bonds with the binding site
❖ Clustering of polar amino acid side chains alters reactivity
 Lecture 5 Protein Function Cont

Evolutionary Tracing
❖ Evolutionary Tracing used to identify sites in protein that are most crucial to the domains
functions
➢ Sites crucial to protein functions don’t change much
■ Ligand binding site
➢ Properties of the binding site could change

Protein binding Interfaces

❖ Surface- String: surface of a protein interact with chain of polypeptide
❖ Coiled- coil: 2 helices wrapped around each other
❖ Surface- surface: most common, 1 fit like a puzzle to the other

Antibodies
❖ Protein binds to very specific molecules
❖ Antibodies (immunoglobulins): proteins produced by the immune system in response to
foreign molecules
❖ Antigen: an antibody’s target
❖ We need to create a lot of different antibody
❖ By modify the binding site (change the amino acid sequence there

Enzymes
❖ Vary in involvement of reaction
➢ DNA polymerase: form chain of DNA and add nucleotides together, not just
binding
➢ Enzymes bind to substrate and convert them (chemically modified)
■ But enzyme unchanged
❖ Catalysts: allow reactions to happen (accelerated), break or make covalent bond in a
controlled way
❖ Crucial to maintain & create the cell, making life possible

Enzymes are grouped based on functions

❖ Hydrolases: cleave reactions
➢ Nucleases: cleave the bond between nucleotides
➢ Exonucleases: break down the bond from the end
➢ Endonucleases: cut in the middle

❖ Proteases: break peptide bonds between proteins

 ❖ Ligase: stick things together
➢ DNA ligase: seal gap between DNA

❖ Kinases: phosphorylate, add phosphate groups

❖ Phosphatases: remove phosphate groups

❖ ATPases: ATP to ADP

❖ GTPases: GTP to GDP

Enzyme Catalysis
❖ 1.Starts with substrate binding
❖ Enzyme + substrate -> ES -> EP -> Enzyme + Product
❖ Increased number of substrates -> increased the rate at which product is formed,
eventually reaching a maximum value (maximum rate) = Turnover number
❖ As substrate concentration increases, the rate of reaction also increases
❖ Km = substrate concentration when at half of maximum speed
➢ Low Km = the binding between enzyme and substrate is strong
➢ High Km = the enzyme- substrate bond is weak

Transition States
❖ When 2 molecules react -> increased local concentration of substrate at the catalytic state
❖ Some of the binding energy contributes directly to the catalysis
❖ Substrates pass through intermediate states of altered geometry and electron distribution
before forming the products of the reaction
❖ Transition state: the most unstable intermediate state
❖ The energy required to get to that unstable intermediate state: Activation energy
➢ The major determinant of the reaction rate (the rate of reaction increases if the
activation energy decreases)

How enzymes speed up reactions?

❖ Active sites of an enzyme contains positioned atoms that speed up a reaction by using
charged groups to alter the distribution of electrons in substrates and the shape of the
substrates (covalent bond between substrates and enzyme’s side chains, those side chains
on enzymes will revert back to its original state)
❖ Those changes in geometry and electron distribution drive the substrate toward the
transition state.
Lysozyme
❖ Act as a natural antibiotic
❖ Cut bacteria cell wall
❖ Catalyzes hydrolysis (add a molecule of water to break bond)
➢ Can only do this when polysaccharide is twisted into intermediate state shape
(altered geometry & electron distribution)
➢ Enzyme distorts the electron configuration of sugar so water can come in
➢ Enzyme will reverts back to its original state
❖ Energy barrier: colliding water molecule can break a bond only of the atoms are in a
transition state

Coenzymes
❖ Enzymes use other non-protein molecules to perform functions that are difficult for
amino acids alone
➢ Ex: carboxypeptidase cuts polypeptide chain, use zinc ions to form bond with
substrate
❖ Enzymes frequently have a small molecule or metal ion associated with their active site
that assist with catalytic function.

❖ The efficiency of enzymes in accelerating chemical reaction is crucial to the maintenance

of life
❖ The rate of reaction in cells are rapid because enzyme catalysis is so effective
❖ Diffusion-limited: the factor that limits the reaction = frequency with which the enzymes
collides with its substrate

❖ Amount of product depends on concentration of

❖ 1.Enzyme
❖ 2.Substrate

Increase reaction rates without increase substrate concentration

Multienzyme Complexes
❖ The cell can increase reaction rate without raising substrate concentration by bringing the
various enzymes involved in a reaction sequence together to form a large protein
assembly
❖ Multienzyme Complexes: enzyme A & B exists in close proximity together during chain
reactions
Intracellular Membrane System
❖ Segregate particular substrates & enzymes that act on them into the same membrane-
enclosed compartment
The Cell Regulates Enzyme Activity
❖ 1.Regulate the expression of the gene that encodes the enzyme
❖ 2.Confining enzymes to particular subcellular compartments
➢ Intracellular membrane system
❖ 3.Protein Destruction
➢ Proteases
❖ 4.Covalently modified to control their activity
➢ Kinases: add a phosphate group
❖ 5.Molecule binding
➢ Enzyme binds to other molecule that’s not a substrate
➢ Small molecule binds at a regulatory site outside the active site
■ Alter the rate at which the enzyme converts substrates to products

Feedback Regulation
❖ Positive regulation: end product stimulate production of that product
❖ Negative regulation: end product stop or slow down production of that product
 Lecture 6: Protein Function Cont
Allosteric Enzymes
❖ Enzymes with 2 & more binding sites
➢ One binding site can be the for substrate ,
➢ the other site can bind to a regulatory molecule
■ helps regulate that enzymes activity
■ can be both positive and negative feedback
❖ Active site: recognize substrates
❖ Regulatory site: recognizes the regulatory molecules
➢ Regulatory molecule binding can change the conformation of active site so it can
either bind or not bind to substrates
Linkage:
❖ 2 proteins that have an affect (positive or negtive on each other
❖ Consider:
➢ protein binds glucose and another molecule X
➢ If binding site X changes as a result of glucose binding, the two binding sites are
coupled
■ The two sites work together, positively regulate each other’s binding
■ Both molecules need to be bound to be
fully activated
❖ When 2 ligands prefer to bind to the same conformation,
it’s called cooperative binding (also called positive
regulation)
➢ Binding of 1 increases the affinity/ability of
binding the other
❖ Negative regulation: the binding of 1st ligand discourages
the binding of 2nd ligand, wants different conformation
➢ still coupled but not cooperative
➢ both molecules prefer different conformations
❖ Cooperative Allosteric transitions:
➢ Consider:
■ Protein with multiple subunits, enzyme is on
■ Binding of inhibitory molecule at one site changes the conformation on
the other subunits, making it easier for another inhibitory molecule to
come in and shut off the enzyme
■ Multiple subunits enable easy transition
compared to just one subunit, so it is
more effective (like peer preasure)
Compartmentalization of Cells: Overview
❖ Eukaryotic Cells are divided to functionally distinct, membrane- enclosed
compartments called organelle
➢ Proteins in each organelle catalyze the reaction that occurs there & transport
small molecules into & out of compartments
➢ Specialized cells rely heavily on different organelles on different extents
❖ Nucleus:
➢ has the genome
➢ the primary site of DNA & RNA synthesis
➢ double membrane + disordered regions
❖ Cytoplasm: (cytosol)
➢ surrounds the cytoplasmic organelles,
➢ site of protein synthesis and degradation (distruction)
➢ not an organelle
❖ Endoplasmic reticulum & ribosomes:
➢ rough ER- has ribosomes, smooth ER- no ribosomes. ER- double membranes
➢ share this membrane with the outer membrane of nucleus
➢ free ribosomes bind to MRNA transcript to make proteins,
■ which can signal where they are needed, those free ribosomes will get to
the er and finish making that protein in that er
❖ Lysosomes:
➢ digestive enzymes that distroy dysfunctional intracellular organelle & particles
brought in from the outside by endocytosis.
➢ Endosomes: fuse with lysosomes
❖ Golgi apparatus:
➢ receives proteins from ER and sends them to various destinations
➢ sorting station
❖ Mitochondria:
➢ Protein import, and energy conversion
➢ generate ATP that cells use to drive reactions & required an input of free energy

Family of Organelles
1. Nucleus & cytosol
a. Communicate with each other through nuclear pore complex
2. Organelles in the secretory & endocytic pathways
a. Moving stuff away or towards
b. ER, Golgi, transport vesicles, endosomes, lysosomes
3. Mitochondria
4. Chloroplast [plastids] (in plants only)
a. Energy conversion in plant
Means of Transport
1. Gated Transport
a. Active transport of proteins & RNA move between the cytosol & nucleus through
nuclear nuclear pore complex in the nuclear envelope (act as selective gates)

2. Protein translocation
a. Transmembrane protein translocators directly
transport proteins across a membrane from
cytosol
b. Exist on the membrane and help transport
proteins across the membrane of different
organelles
c. Snakes through

3. Vesicular transport
a. Membrane- enclosed transport- ferries proteins from 1
compartment to another
b. Protein targeting: using signal sequences to get proteins
to its destination in the cytostome, cytsomal protein has
no signal sequence
Cell address:
 Where the protein needs to be (signals via the signal sequence)
 Signal sequences (sorting signal) -sequences on the N-terminus or in the middle of the
polypeptide chain
 Signal peptidases = can remove the signal sequence from the finished protein once the
sorting process is complete
 Each signal sequence specifies a destination
o Ex: If protein needs to be transported to ER -> signal sequence on N- terminus (5-
10 hydrophobic AA), known as N- terminus ER signal
 Protein Targeting: Using signal sequences to get proteins to their destinations

Sorting receptors
❖ Signal molecules are recognized by their complementary sorting receptors that guided
proteins to their destinations, usually the receptor will go back leaving only the protein
❖ If a protein needs to be transported
➢ Across a membrane, translocator recognizes sorting signal
➢ To a vesicle or organelle, complementary receptor in the membrane must
recognize the sorting signal
❖ The receiving organelle also need to recognizes the sorting signal
The ER
❖ ER membrane continuous with other membranes
❖ ER ribosomes
❖ Proteins destined for ER are made while being transported to the ER (rough ER)
❖ ER structure:
➢ rough ER, smooth ER, and transitional ER (buds off and goes to the Golgi)

❖ Co- translation:
➢ Finishing the protein but also translocating it
➢ most cells import proteins into the ER before completing polypeptide chain
synthesis
❖ Ribosome that is synthesizing the protein is attached directly to the ER membrane
❖ Post- translation:
➢ Regular translation
➢ cytosolic ribosomes complete the synthesis of protein and
release it before translocation
■ If it doesn’t have the ER signal sequence, proteins
are completely made, and then transported to the
proper organelle

ER Signal Sequences
❖ ER captured 2 types of protein from the cytosol
➢ 1.transmembrane protein: exist in the membrane
➢ 2.water soluble proteins : go past the membrane and into the lumen
❖ Directed to ER membrane by an ER signal sequence
➢ Then it is cleaved off by a signal peptidase in the ER membrane
❖ Not all the proteins that enter the ER are ER Resident Proteins (specific signal sequence)
➢ No ER resident signal
■ Use ER as a first step (Secretory pathway)
 Lecture 7: ER
The Signal Hypothesis
❖ How protein transport works in ER
❖ Small & large ribosomal subunits come together during protein
synthesis
➢ the subunits are bind to MRNA transcript

Signal Recognition
❖ ER signal sequence is guided to the ER membrane by 2 components
➢ 1.Signal Sequence Particle (SRP)
➢ 2.Signal Receptor (recognizes SRP)
❖ ER signal sequence (hydrophobic region) has at least 8 nonpolar amino acid chain at its
center
❖ SRP’s signal sequence binding site = large hydrophobic signal sequences lined by
methionines (flexible side chains, can accommodate a wide variety of Signal sequences),
➢ allowing SRP to bind to a variety of signal sequences

❖ SRP binds to signal sequence on two sites: ER signal sequence and

blocks elongation factor binding site
➢ Temporary stops it

❖ Why block?
➢ 1.Transient pause of protein synthesis
■ Reduces the risk of the protein completely being
finished before it reached the ER
➢ 2.Ensures that large portions of a protein that could fold
into a compact shape are not made before reaching the translocator in ER
membrane
Signal + SRP directs ribosome to ER membrane

1. Signal sequence recognized by SRP, blocked translation

2. SRP binds to its receptor
3. SRP receptor brings the complexes to a translocator in the ER membrane
4. SRP + receptor released together
5. SRP + receptor disassociates, SRP goes to cytosol while receptor stays in the ER
membrane
6. Translation continues and translocation begins in the ER membrane

Ribosomes
❖ 2 types of ribosomes (functionally and structurally the same)
➢ Membrane-bound ribosomes
➢ Free ribosomes
❖ Polyribosomes: many ribosomes bound to a single mRNA molecule
➢ Sometimes you need multiple proteins being made from the same transcript
■ Make multiple copies at the same time
❖ Free Polyribosomes, moves from 5’ end to 3’end
❖ Membrane bound Popyribosomes
Translocator
❖ The polypeptide passes through a water- filled channel in the ER membrane translocator
❖ Core of the translocator = Sec61 Complex
❖ Hydrophilic region can come down through the core while the
❖ hydrophobic ER signal sequence enters and leaves the translocator laterally.

Post- Translational Translocation to ER

❖ ER translocator needs accessory proteins that feed the
polypeptide chain into the pore and drive translocation
➢ Ex: BiP- Binding Protein associates with Sec61 ATP-
dependent cycles
❖ Catalyzes hydrolysis of ATP to ADP, energy released used to
pull the protein through
❖

Translocon = translocator + accessory proteins

Start Transfer Peptide

❖ ER Sequence recognized by the binding site
on sec61 complex along the lateral surface
of the hydrophobic core of bilayer, serves as
a start- transfer sequence
❖ **signal peptidase chops signal sequence off
after translocation, releases latterally
Single- Pass Transmembrane Protein
❖ Method 1: stop- transfer signal -> proteins goes into the ER lumen until reaching this
sequence
➢ ½ is in the lumen, the other ½ is in the cytosol
❖ Method 2: When the start- transfer sequence is in the middle, it will release the protein
and ER sequence in the ER membrane
➢

Multi- pass Transmembrane Protein

❖ Protein passes multiple times back and forth between the lipid
bilayer, multiple start and stop transfer signal
❖ Internal signal sequence not going to be cleaved
ER Resident Proteins
❖ Some ER proteins reside there-- contain an ER retention signal
❖ BIP: pulls protein into the ER through the Sec61 ER translocator

Protein Quality Control

❖ Proteins in the er have:
➢ Addition of oligosaccharides to proteins
■ String of monosaccharide’s
❖ Protein glycosylation: adding of the oligosaccharides to the protein
➢ Added to the amino group of the asparagine
■ Can be categorized as asparagine linked/N-linked
➢ precursor oligosaccharide is transferred to the NH2 group of an asparagine in the
protein-- it is said to be N-linked [Asparagine- linked]
❖ Catalyzed by: Oligosaccharyl Transferase (Glusosyl Transferase)
Why Oligosaccharides?
❖ Protein quality control check
❖ Some proteins needs N- linked glycosylation for proper folding in the ER
➢ ER Chaperone Proteins:
■ Calnexin
■ Calreticulin
● Are lectins: Bind to carbohydrates
■ Bind to oligosaccarides on incompleatly folded proteins and retains them
in the ER
■ High affinity for oligosaccharides, bind to them and prevent protein from
misfoldisng

Oligosaccharide Processing

❖ ER Glucosidases catalyze glucose trimming, removing glucoses one-by-one

❖ One glucose remains because lectin’s high affinity for glucose
Protein Quality Control
1. Glucose trimming by glucosidase
2. Calnexin binds to glucose
3. Glucosidase removes the last glucose from oligosaccharide, protein leaves calnexin
4. Protein tries to fold itself
5. Glucosyl Transferase determines if protein is folded correctly, if not it will add a glucose
back to the oligosaccharide and start the cycle again. Otherwise protein exits the ER.
a. Gives the protein another chance to fold properly

Glucosidase removes glucose, glucosyl transferase adds glucose on

 Lecture 8: ER & Intracellular Membrane Traffic
Retrotranslocation
❖ Misfolded protein given multiple chances to fold correctly
➢ But they don’t keep trying forever
➢ N- linked oligosaccharide determines how long protein
stays in that cycle
➢ Serve as timers
❖ Mannosidase will remove the mannose on the oligosaccharide
before glusosyl transferase can add a glucose back to the
oligosaccharide
❖ Proteins that fold and exist the ER faster than the Mannosidase can remove its target
mannose and escape degradation
➢ You just want to fold properly and get out, the longer you stay the more likely it is
that the mannosidase will mark you for destruction
❖ After mannose is taken off, misfolded proteins can be exported from the ER back to the
cytosol and destroyed in proteasomes: This is called retrotranslocation
❖ How do proteasomes know which protein to degrade?
➢ They leve through a specific translocator
➢ The misfolded protein leaves through translocator complex with E3 Ubiquitin
Ligase Enzyme that attaches Polyubiquitin tags (like a bag tag) to the folded
proteins as they emerge into cytosol-- marking them for destruction
The process of Retrotranslocation
1. Misfolded proteins form sulfide- sulfide bond, disulfide isomerase try to prevent sulfide-
sulfide Bond.
2. E3 ubiquitin ligase enzyme adds Poly ubiquitin tags to the misfolded protein.
3. Proteases (enzyme) in proteasome chop up misfolded
proteins in the cytosol
4. AAA- ATPase, accessory enzyme hydrolyzes ATP to
ADP, helps to pull the protein through the ER membrane
a. In this case the energy released by hydrolysis
helps pull the protein through the membrane
5. N-glycanase: saves the oligosaccharide
Unfolded protein response
❖ When the number of misfolded protein increases in concentration
❖ Heat shock response: increase transcriptions of chaperone proteins
❖ Unfolded protein response: increase transcription of genes code for chaperones,
retrotranslocation, protein degradation, ER chaperones and other proteins that increase
protein- folding capacity
Intracellular membrane traffic
❖ Endocytosis : pinching in of plasma membrane, form endosomes.
➢ Ways in which nutrients and vitanmines can be move inwards

❖ Endocytic Pathway: Leads inwards from the plasma membrane

❖ Exocytosis: delivers new proteins to plasma membrane and extracellular space
❖ Secretory pathway: leads outwards from ER towards Golgi to plasma membrane

➢
Transport vesicles
❖ Lumen: interior space of each membrane- enclosed compartment
➢ proteins can travel in the lumen, they are transported from one compartment to
another compartment by transport containers called vesicles.
❖ Budding -> fusion
❖ Transport vesicles contains cargo:
➢ Can move membrane components and soluble luminal molecules

 must be selective
o 1. only take up appropriate molecules
o 2. only fuse with appropriate Target molecules
 molecular address: displayed on cytosolic surface of membranes
 they need to recognize what is coming in
 Specific combination of molecular address combinations matter
❖ Coated Vescicles
➢ vesicles has two coats
■ The inner coat selects the cargo
■ Outer coat gives it its shape
➢ Before they fuse with target membrane the discard their coat
Three types of coats
❖ 1. Clathrin- coated: golgi to plasma membrane
❖ 2. COP1- coated: Golgi to ER
❖ 3. COPII- coated: ER to Golgi
Clathrin- coated vesicles
❖ Golgi to plasma membrane
❖ Outer coat = Clathrin, major protein component of the coat
➢ Made up of 3 large subunits and 3 small subunits
❖ Called Triskelion, assembles into a basketlike framework of hexagons & pentagons to
form coated pits (buds) = outer layer

❖ Adaptor proteins = Inner Coat

➢ binds the clathrin coat to the membrane and
trap various transmembrane proteins (cargo
receptors), help selects targets
❖ Adaptor proteins are specific to different receptors
and specific cargo molecules
❖ Adaptor protein bound to receptor, receptor is
bound to cargo
❖ Adaptor proteins select a specific set of
transmembrane proteins + the soluble proteins that
interact with them and package them into newly
formed clathrin-coated transport vesicle

PIPs
❖ Specific adaptor protein
❖ PIPs that can be phosphorylated in different regions, which
alters the adaptor protein’s conformation, exposing binding sites
for cargo receptors in its membrane.
Membrane- Bending Proteins
❖ Have crescent- shaped domains (BAR Domains)
➢ Help bend the membrane into the bud shape by binding to the membrane
➢ Impose their shape on that membran, helping them form that circular bud

❖ Dynamin: cytoplasmic protein that tethers to the membrane,

➢ helps tether the neck of the bud and recruit other proteins
to seal off the vesicle
Release from Membrane
❖ As soon as phosphorylated PIP leaves, the adaptor proteins bond is weakened, so the
adaptor proteins also leave the cargo receptor
➢ The coat disassembles
❖ Uncoating ATPase: ATP hydrolysis to help get rid of the coat

Controlling Coat Assembly: only assemble when and where they are needed
❖ Method 1: PIPs plays a major part in regulating the assembly of clathrin coats
❖ Method 2: GTPases control coat assembly
❖ Coat- recruitment GTPases are members of the family GTPases
➢ Hydrolyze GTP -> GDP
➢ Releases a lot of energy
❖ GTP- Binding Proteins:
➢ proteins that bind to GTP/GDP
❖ Molecule switches:
➢ Switch between GTP to GDP
➢ flip between the active to inactive
■ protein bound to GTP, it is active
■ protein bound to GDP, it is inactive
➢ 2 classes of molecule switches:
■ Guanine Nucleotide Exchange Factor (GEFs):
● GDP -> GTP (Activating the protein/ turn them on)
■ GTPase activating proteins (GAPS):
● GTP -> GDP (Inactive the protein)
❖ GTPases : GTP binding proteins
➢ Coat recruitment GTPases include
■ ARF Proteins: assembly of COP1
and Clathrin coat assembly at
Golgi
■ Sar 1 Proteins: assembly of
COPII at ER membrane
 Lecture 9: Intracellular Membrane Traffic Cont:
COPII- coat disassembly
❖ Vesicles pinches off but sealed coat stays on until it docks at target membrane
❖ At target membrane, kinases phosphorylate the coat proteins
❖ Coat disassembles and vesicle ready for fusion
➢ Both Clathrin and COPII (assembled at Golgi) disassembles as soon as the
vesicles pinches off
COPI - coat disassembly
❖ Shed coat after they pinch off
❖ The curvatures approaching that of a transport vesicle serves as a signal to recruit ARF-
GAP.
➢ GAP then inactivates ARF (GTP to GDP) and coat disassembles.
Vesicle budding
❖ Budding occur where intracellular membranes are already curved
❖ Oversized cargo:
➢ bind to transmembrane packaging proteins in the ER which control assembly of
COPII coat components to accommodate the oversized cargo
Specificity in targeting
❖ All transport vesicles display surface markers
➢ that identify them according to their type of cargo and origin
❖ Target membrane display complementary receptors
➢ that recognize the appropriate markers
❖ Rab proteins are GTPases that help bring vesicles to target membranes
➢ Organelles have at least one Rab protein on their cytosolic surface
➢ Can function on transport vesicles and/or target membranes
❖ When it is GDP bound (Rab-GDP) they are inactive
➢ Rab-GDP binds to GDI (Rab-GDP Dissociation
inhibitor)
■ It keeps them soluble in cytosol
❖ Membrane bound Rab-GEFs activates Rab proteins
➢ Rab-GDP to Rab-GTP
❖ When GTP is bound (Rab-GTP) they are active and can anchor to the membrane

❖ Rab Effectors
➢ Rab-GTP binds to other proteins called Rab Effectors- mediators
of vesicle transport, membrane tethering and membrane fusion
➢ They are tethering proteins that have long, threadlike domains that
can link two membranes together.
■ Allows for fusion to happen
 ❖ Rab Activation
➢ 1.Rab-GDP/GDI complex encounters a Rab-GEF
■ GEF activates proteins
➢ 2.GDI is released & Rab-GDP is converted to Rab-GTP
➢ 3.Active Rab-GTP becomes anchored to the membrane and
recruits more Rab to the site: Rab cascade
❖ Rab Cascade
➢ Ordered recruitment of sequentially acting Rab proteins is called a Rab Cascade

❖ SNARE proteins
➢ helps vesicles to fuse with target membrane in order to dump its cargo
➢ Once tethered to target membrane, vesicle needs to unload cargo by membrane
fusion
➢ Membrane fusion = bringing lipid bilayers close to each other so they can merge
■ v-SNAREs: found on vesicle membranes (1 protein)
■ t-SNARESs: found on target membranes (3 proteins)
● v-SNAREs & t-SNAREs have helical domains that will wrap
around each other to form a very stable trans-SNARE complex
● 2 v-SNARE 3 t-SNAREs= 1 set, a molecule can have mutiple sets
The Process of Fusion
1. Rab protein is active
2. Rab proteins in vesicles and the surface of target
membrane needs to recognize each other
3. Rab effector tethers vesicle to target membrane
4. v-SNAREs on vesicle & t-SNAREs on
membrane form trans-SNARE complex, locking
2 membranes together for fusion
5. Interactive SNAREs need to be pried apart before
they can function again. NSF proteins work with
accessory proteins, using the energy from ATP to ADP (hydrolysis) to pry apart the
trans-SNARE complex.

Transport from ER to Golgi

❖ Some transport vesicles take cargo molecules to next compartment in pathway
❖ Some retrieve escaped proteins and return them to a previous compartment
➢ 2 way transport
❖ Proteins Leaving ER
➢ Proteins leave the ER towards Golgi or beyond in COPII coated transport vesicles
➢ These vesicles bud from specialized regions of the ER called ER exit sites
 ➢ The cargo membrane proteins display exit signals (transport signals) on their
cytosolic surface adaptor proteins of inner COPII coat recognizes these
➢ Soluble cargo proteins (in the ER lumen) have exit signals that attach them to
transmembrane cargo receptors
❖ Proteins leaving the ER
➢ need to be properly folded,
➢ If they are misfolded, they need to bind to chaperone proteins
➢ The chaperones may cover up exit signals or anchor proteins in ER.
➢ Failed proteins are then transported back into the cytosol and degraded by
proteasomes.

❖ Vesicular Tubular Clusters

➢ Vesicles can fuse together to form those
clusters with SNARE-proteins, they can link
not only membranes but also vesicles
Retrieval (retrograde) Transport
❖ Proteins need to get back to the ER from Golgi- ER resident proteins

❖ Returning escaped proteins back to the ER depends on: ER retrieval signals

➢ Incase they are accidently sent out, they have a “homing signal”
❖ Soluble ER resident contain a short ER retrieval
signal at the C-terminal: KDEL Sequence
recognized by KDEL Receptor (KKXX sequence on
transmembrane proteins)
1. KDEL receptor
a. low affinity for ER resident proteins at ER
b. High affinity for ER resident A other places
i. Involved in helping bring resident proteins back
2. At vesicular tubular cluster or Golgi apparatus, ER resident proteins can be saved by
KDEL receptor (high affinity for ER resident protein now)
❖ Only those proteins that escape are captured and returned via KDEL receptors
❖ ER resident proteins remained in the ER via Retention mechanism:
➢ ER resident proteins bind to each other and form complexes that are too big to
enter transport vesicles efficiently
❖ Likewise Golgi enzymes that function together also bind to each other so they don’t leave
the Golgi apparatus
 Lecture 10
Golgi Apparatus:

❖ Proteins enter the Golgi from CGN (Cis Golgi Network)

❖ Proteins leave the Golgo from TGN (Trans Golgi Network)
❖ Sorting happens at the TGN

Oligosaccharide Chains
❖ The cell reuses oligosaccharides for new functions in the Golgi
➢ to generate the heterogenous oligosaccharide structures as the proteins proceed
through CGN to TGN.
❖ Glycosylation: the adding of oligosaccharide chains to proteins in Golgi
➢ By the time it gets to the trans cisternae, the glycosylation is complete
❖ Two classes of N- linked oligosaccharides are attached to mammalian glycoproteins
➢ 1.Complex oligosaccharides- when original N-linked oligosaccharides added in
ER is trimmed and further sugars are added back to the golgi
➢ 2.High-mannose oligosaccharides- trimmed but have no new sugars added to
them
Golgi transport hypothesis
1. Cisternal Maturation Model: different part of cisternae
mature into each other by acquiring and losing certain
proteins
a. Different cisternae mature into the next
b. This is how things ar emoved across thecisternae

2. Vesicle Transport Model: transport happens in form of budding

and fusion of vesicles
❖ Retrograde transport happens regardless of the hypothesis
❖ Support for both of those hypothesis: core of long-lasting cisternae
might exist at center of each Golgi cisterna, regions at rim may
undergo continuous pinching off & fusion

Trans Golgi network & Lysosome

❖ Secretion
❖ Separate into 3 classes of proteins before leaving the TGN:
➢ 1.Destined for lysosomes
➢ 2.Immediate delivery to cell surface
➢ 3.Destined for secretory vesicles
■ 2+3: exocytosis
Lysosome
❖ Lysosomes: membrane enclosed organelles filled with enzymes that digest
macromolecules (proteases, nucleases, etc)
➢ The point is to digest macromolecules
❖ These enzymes are acid hydrolases: they are hydrolases that work best at acidic pH
➢ That is why the ph in lysosomes is 4.5-5
➢ Cytosol has a ph of 7
❖ Transport proteins in the lysosome membrane carry the final products of digestion (such
as AA) to cytosol where cells reuse or excrete them.
❖ Lysosomal hydrolases: (Protein): enzymes are delivered to the endosome in transport
vesicles that bud from the TGN.
The process of digestion in lysosome
❖ Late endosomes fuse with lysosomes to form
endolysosomes.
❖ Lysosomal hydrolases then destroy the intraluminal
vesicles & proteins in it.
➢ Results in a nice lysosome
❖ How are lysosomal hydrolases selected in the TGN
➢ They have unique marker in the form of mannose 6-phosphate (M6P groups)
■ Tag says that they need to get to the lysosome
■ It’s a phosphorylated mannose
➢ Signal Patch: AA sequence with info about the destined location of protein
Transmembrane M6P Receptors
❖ Present in TGN
➢ 1.Recognizes the M6P groups
➢ 2. Binds to the lysosomal hydrolases on the lumenal side of the membrane and to
adaptor proteins in assembling clathrin coats on the cytosolic side.

1. Addition of Phosphate to mannose on

lysosomal hydrolase ---> M6P
2. At TGN, the M6P is recognized by the M6P
receptor
3. Transport to endosome, clathrin coat sheds
off
4. At the early endosome, M6P receptor is
retrieved by retromer coat.
5. Lysosomal hydrolase dissociate at acidic pH
6. Removal of phosphate
7. Early endosome matures to late endosome and fuse with lysosome to form endolysosome
Exocytosis: fusion of the vesicles with the plasma membrane
1. Constitutive Secretory Pathway: operates continuously,
a. immediate delivery to cell surface.
b. Default pathway ( doesn’t have a specific
signal)

2. Regulated Secretory
Pathway: soluble proteins and other substances are stored in
secretory vesicles until they are needed for later release by
exocytosis
i. those vesicles are known as dense-core granules
ii. calls that are specialized for secretion of some of
their products RAPIDLY

❖ Most of the membrane of the secretory vesicles that leave the TGN is only loosely
wrapped around the clusters of secretory proteins
❖ The immature secretory vesicles matures and there are less space in the vesicles, proteins
packed them super close to each other, this allows cell to store a lot of things together and
release them all at once

❖ Secretory vesicle fuses with plasma membrane & its contents are discharged
❖ Membrane becomes part of the plasma membrane
➢ This increases the surface area of the plasma membrane but only transiently
➢ Endocytosis and exosytosis is happening at the same time so the plasma
membran doesn’t increase
❖ Membrane compounds are removed from the surface by endocytosis as fast as they are
added by exocytosis
 Lecture 11: Endocytosis

Endocytosis
❖ Process by which the cells take up plasma membrane components, fluid, solutes,
macromolecules, substances
❖ Plasma membrane pinches in to form endocytic vesicles to take in stuffs
❖ 2 types of Endocytosis
➢ 1.Pinocytosis: the material to be ingested is enclosed by a small portion of the
plasma membrane, then pinches off and forms endocytic vesicles
■ “cell drinking”
➢ 2.Phagocytosis: a special kind of pinocytosis in which large endocytic vesicles
called phagosomes are fused to take up large particles on demand
■ “cell eating”
Endocytic pathway
1. Vesicles formed and pinches in
2. Endosome maturation: early endosome
matures into late endosome
3. Things from outside of cells can also enters
the endolysosome pathway
4. Retrograde transport: cell stop recycling at
one point and start to destroy things, but
things can also be sent back from lysosome to
TGN
Pinocytosis
❖ All eukaryotic cells continually ingest portions of their plasma membrane in the form of
small pinocytic (endocytic) vesicles
❖ Cell’s surface area and volume remain unchanged during pinocytosis because the same
amount of membrane being removed by endocytosis is being added to cell surface by the
exocytosis-> Endocytic-Exocytic Cycle
Receptor- Mediated Endocytosis
❖ Transmembrane receptors, accumulate in these pits and enter the cell in clathrin coated
vesicles
❖ Ex: Cholesterol uptake: Cholesterol is transported in the blood in the form of LDLs (Low
Density Lipoproteins)
 ❖ All of the Receptor-Mediated Endocytosis uses clathrin-dependent- internalization routes
❖ Guided to clathrin-coated pits by signals (phosphoralized pips) in their cytoplasmic tails
that bind to adaptor proteins in the clathrin coat
❖ Some enter coated pits regardless of whether they have bound their specific ligands

Fate of Endocytosed Receptors

1. Returned to the plasma membrane
 Early endosomes = sorting stations in the endocytic pathways
 Endocytosed ligands (situational specific)
o 1.Dissociate from their receptors
o 2.Remained bound to their receptors
 Ex: transferrin receptors: protein that carries iron in the blood
 low ph of endosome allow release of irons, not transferrin
 Apotransferrin: iron-free transferrin
 Receptor and the apotransferrin is taken back
2. Receptor Down Regulation
❖ Destroy receptors
➢ Receptors marked for destruction are marked with Monoubiquitylation
(multiubiquitylation)
➢ Ubiquitin tagging for sorting into the clathrin- dependent endocytic pathway
■ Don’t want these vesicles to remain on the membrane
❖ Ubiquitin binding protein recognizes the attached ubiquitin and helps direct the modified
receptors into clathrin coated pit
➢ Recognize and sort the ubiquitylated receptors into intraluminal vesicles
➢ Addition of ubiquitin blockers receptors from entering the recycling pathway
(returning to the plasma membrane)
■ Eg: EGF: Epidural Growth Factor
● Receptor Down Regulation: gets rid of receptors until needed

Endosome Maturation
❖ As endosomes mature, patches of their membrane pinch off to form
intralumenal vesicles
❖ The entire vesicle with those intralumenal vesicles = multivesicular body

❖ Early endosomes have 2 domains:

➢ 1.Tubular Domain: rperipheral egions where vesicles will pinch off and return or
fuse with cargo brought in
➢ 2.Vacuolar Domain: center of early endosome and will remain there throguout
maturation process
 1. Endosomes change shape and location
a. Fusing and moving toward the interior of cells
2. Molecular makeover
a. The contents are changing (receptors taken back)
3. Acidifies the endosome lumen
a. Lumen becomes more and more acidic because lysosomal proteins need acidic
environment to destroy things
4. Stop receptor signaling activity
a. Use endosome maturation to silence receptors
5. Lysosome proteins are delivered from the TGN to the maturing endosome

Endocytosed Proteins -> Multivesicular Bodies

❖ When receptors tagged with ubiquitin tags, endosomes pinches in and form intralumenal
vesicles to get receptors into lysosome for degradation
❖ If receptors are on the membrane of late endosome, they won’t be destroyed
➢ If it needs to be destroyed they have to be a part of the intraluminal vesicles
❖ Once the pinching in initiated ubiquitin tags will be recycled

Intralumenal vesicle formation

❖ At the endosomal membrane, the ubiquitin tags are recognized again by cytosolic ESCRT
protein complexes: bind and mediate the sorting process into intralemenal vesicles
❖ PIP: (phosphorylated) served as a docking site for ESCRT at the membrane , so escrt can
binds to ubiquitin tags on the receptors
Recycling Endosomes
❖ Recycling: most receptors are recycled and returned to the same plasma membrane
domain from which they came
❖ Transcytosis: receptors are recycled and return to a different domain of the plasma
membrane

Fates for Transmembrane Receptor Proteins

1. Recycling
a. Receptors get abc to the plasma membrane or use
recycling endosomes
2. Transcytosis
a. Goes back to a different region on the plasma
membrane
3. Degradation
a. Receptors taken to degradation in endolysosome

Cell can regulate the release of membrane proteins from recycling endosomes
❖ Intracellular signals to increase rate of receptors on the plasma membrane, so they can
take in that certain molecules

Phagocytosis: specialized form of endocytosis

❖ Phagosomes = large endocytic vesicles used to ingest large particles
❖ Macrophages = cells that detects, engulfs and destroys invades
❖ Neutrophils = type of white blood cells
➢ Protects from infections

Process of Phagocytosis
1. Bacteria coming in, phagosome took in bacteria
2. Phagosome fuses with lysosome to form
phagolysosome
3. Bacteria destroyed in phagolysosome and soluble
debris released through exocytosis
 Lecture 12
Transport into Mitochondria
❖ They specialize in ATP synthesis

❖ Mitochondrial proteins are first fully made as mitochondrial precursor proteins in the
cytosol then translocated into mitochondria via a post-translational mechanism
➢ Not cotranslational
➢ Proteins entering the matrix space: have a signal sequence at their N-terminus
that a signal peptidase removes after it gets to its destination
❖ Membrane proteins have internal signal
sequences that are not removed
❖ Mitochondria signal sequence forms an alpha
helix with the positively charged amino acids
on one side and the hydrophobic ones are on
the opposite side

Protein Translocators
❖ Multi-subunit protein complexes that mediate protein movement across the mitochondrial
membrane
➢ TO(outer)M complex: transfers proteins
across and into the outer membrane
➢ TI(inner)M complex: transfers proteins
across the inner membrane
■ TIM23: transport soluble protein
in the matrix, can also insert into
the inner membrane
■ TIM22: help move proteins
involved in ATP & ADP
■ TOM and TIM acts as receptors for mitochondrial precursor and forms
the translocation channel
➢ OXA complex: help insert inner membrane protein made in mitochondria
➢ SAM complex: help proteins fold properly in the outer membrane
➢ Those complexes act as receptors for mitochondrial precursor and form the
translocation channel
Protein Folding

1. Import receptors of the TOM complex bind the signal sequence

2. Interacting proteins are stripped off
a. Mitochondrial precursor proteins remained unfolded by interacting with
chaperone proteins of the hsp70 family in the cytosol
b. Cytosolic hsp70 chaperone is released from the precursor protein by ATP
hydrolysis
3. Unfolded protein is fed into the translocation channel
4. TOM complex transports the signal sequence across the outer membrane to the
intermembrane space
5. Binds to a TIM complex & opens the channel
a. Mitochondrial hsp70: binds to imported protein chain as it emerges from the TIM
translocator in the matrix space and pull protein across the channel by using ATP
hydrolysis
i. Helps pull the protein through (bind and release)
6. Translocated into the matrix space (or can be inserted to the inner membrane)
7. Protein passed to mitochondrial hsp60 after interacting with hsp70, helps the unfolded
chain fold by binding and releasing it through cycles of ATP hydrolysis.

How SAM helps TOM

- Enters via tom, but sam helps fold it properly
Import to inner mitochondrial membrane & intermembrane space
❖ Method 1: Matrix targeting signal sequence
➢ Hydrophobic stop-transfer signal inserted to the inner membrane and prevent
further translocation

❖ Method 2: Oxa complex helps insert into the inner membrane

➢ Protein coming in from the cytosol and the oxa complex can help inset it into the
intramembrane space

Transport into Chloroplasts

Protein transport in mitochondria vs chloroplast
1. Both processes occur post-translationally
2. Use translocation complexes
3. Requires energy
But the protein components differ!

Protein Transport in Mitochondria

❖ Outer membrane transport,
TOC ( TOM counterpart)
❖ Inner membrane transport ,
TIC ( TIM counterpart)