CELLBIO Exam 1 (Lecture 1-8)

design a experiment (4 steps)

1. question/hypothesis

2. approach/model system

3. design procedure/execute experiment

4. make observation/interpret results

experimental approaches (3)

biochemical

microscopy

genetics

biochemical experimental approach

observe structure/behavior of specific molecules and interactions

• purify/separate biomolecules

• detect proteins with antibodies

• identify interactions between biomolecules

• monitor biochemical reactions

microscopy experimental approach

visualize shape, location, and behavior of organisms, cells, cell parts, and molecules

• observe molecules, cells, tissues, organisms

• detect specific molecules, cell parts

• live imaging → observe dynamics

genetics experimental approach

evaluate function/structure of cell or cell part when DNA is altered

• identify cells/individuals with phenotype of interest and genes

• create mutation in region of interest, evaluate the effect on cells

• alter expression levels of RNA or protein, evaluate effect on cells

• add sequence to gene → adds AAs to protein → fusion protein or tagged version is useful for experiments

amino acid characteristics determined by

sidechain

charged sidechain → polar, uncharged → nonpolar

• negative charge → acidic

• positive charge → basic

N-terminus vs C-terminus

N-terminus (Amino terminus): This is the "start" of the protein chain and contains the free amino group (-NH₂). It's called the N-terminus because it has the nitrogen atom from the amino group. It is the first part of the protein synthesized during translation.

C-terminus (Carboxyl terminus): This is the "end" of the protein chain and contains the free carboxyl group (-COOH). It's called the C-terminus because it has the carbon atom from the carboxyl group. It's the last part of the protein synthesized during translation.

common protein structures

secondary structure

alpha helix - right-handed coiled structure, where the polypeptide backbone twists around an axis, and the side chains (R groups) extend outward from the helix. The structure is stabilized by hydrogen bonds

beta strands - formed by two or more polypeptide chains that align side by side. The backbone of the chains forms a sheet-like structure, where each chain runs in a parallel or antiparallel direction. Hydrogen bonds stabilize the arrangement between the strands.

folded proteins form complexes

monomer - single protein subunit

homodimer

heterodimer

trimer

tetramer

protein structure determines function

function by interaction/binding noncovalently (hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals forces) with other molecules like proteins, lipids, nucleic acids

proteins can be covalently modified by

addition of specifc groups to specific AAs

• glycosylation

• phosphorylation

protein function can be regulated by…

covalent modification or binding to other molecules

model systems

cell or organism commonly used for research as example to understand biology of cell or organism more generally

key characteristics of model systems (5)

• easy to maintain/grow

• can do experimental manipulation (and observe it)

• subject to existing biological data

• existing experimental tools/protocols

• minimal genetic variation in population

choose model system based on (8)

• behavior/features of interest

• tools, technique, biological information available

• timescale

• cost

• ethicality

• applicability to other systems

• simplest that’s sufficient

• better than other systems

cell culture (4)

removed from multicellular organism grown in lab to study cell behavior and experiment outside of organism in vitro

manipulate cell behavior without affecting organism

immortalized cell lines (human tumors) → indefinite replication

some cells are difficult to maintain/grow

microscopy

visualize things not visible to human eye

parts of microscope

beam source - light or electrons

sample - must be prepped properly based on microscope type

objective lens - collect signals that go through sample

detector - generate image

light vs. electron microscopy considerations (4)

determined by sample and features of microscope

• magnification

• detection

• resolution

• contrast

resolution

shortest distance between 2 points/objects distinguished as separate

resolution = r = 0.61λ/nsinθ

lower r = better resolution

• λ = wavelength of beam used

• n = property of media between objective and sample (air, water, oil)

• θ = property of microscope

contrast

difference in signal intensity between object and background

increase contrast by

• manipulating light/electron beam

• manipulating sample by adding stain, fluorescent molecules, or heavy metal

sample preparation steps include (6)

• fixation

• permeabilization

• dehydration/drying

• freezing

• sectioning

• mounting

sample preparation based on (3)

• type of sample

• type of object visualization desired

• type of microscope

Light Microscopy

limit of resolution: 200 nm

alive or dead cells

method of increasing contrast by manipulating sample/light wavelengths are important

types of LM (9 points)

white LM - visible/white light (all λs) → sample → objective lens → detector

• brightfield M - manipulate light path to increase contrast → image formed by the contrast between the sample and the surrounding medium

• phase contrast - manipulate light to visualize light/dark regions → shifting the phase of light passing through different parts of the sample, making it easier to see internal structures.

• Nomarski/differential interference contrast - manipulate light in a different way so image appears 3D → uses polarized light to create 3D-like images of transparent samples. It improves contrast and resolution, making it easier to observe fine details.

fluorescence M - light (specific λ) → sample with fluorescent molecules (excited by specific λ, emit lower energy/longer λ) → object lens (collect lower λ) → detector

different fluorescent molecules have different excitation λs and emission λs, so they can be combined in one experiment

• worst: epifluorescence (widefield) - entire sample illuminated at one time, emitted light used to generate image

• confocal - one thin section illuminated at a time, images combined to form final image

• best: superresolution - only a few molecules illuminated at a time, images combined to form final image, resolution limit <200 nm

approaches to detect protein by fluorescence microscopy

1. Immunofluorescence using fluorescently labelled Ab that binds to protein of interest

2. Immunofluorescence using fluorescently labelled Ab that binds to protein tag attached to protein of interest

3. Direct fluorescence of flourescent protein tag attached to protein of itnrest

Approach 1: Immunofluorescence using fluorescently labelled Ab that binds to protein of interest

1. Fix sample - limit molecule movement, chemicals react with and crosslink proteins to each other to stabilize sample

2. Permeabilize sample - with detergent, disrupts lipids in cell membrane to allow Ab to access proteins

3. Add Ab

4. Detect with fluorescent microscopy

primary Ab - binds to protein of interest

secondary Ab - has fluorescence, binds to primary Ab

Approach 2: Immunofluorescence using fluorescently labelled Ab that binds to protein tag attached to protein of interest

protein tag - specific AA sequence useful since Ab can bind here and be detected/purified, can work on any protein

protein tag + POI = fusion protein

1. Generate fusion protein:

   1. DNA encoding protein tag covalently attached to DNA for POI, fusion can occur outside of cells

   2. Introduce fusion DNA into cells via transfection, electroporation, viral infection, microinjection

2. Fix, permeabilize, add Ab, detect with fluorescent microscopy

Approach 3: Direct fluorescence of fluorescent protein tag attached to protein of interest

• fluorescent protein i.e GFP

• visualize by fluorescent microscopy without any further sample prep i.e fixing

• detect fluorescent protein in live or dead cells

• live cells, visualization of dynamics (since no further sample prep like fixing that kills cells)

Electron Microscopy

• uses electrons as beam source

• electrons very small → resolution possible. down to atomic level

• imaging occurs in vacuum → live cell imaging not possible

• cells fixed/frozen to preserve structures

• heavy metals can be arranged to add contrast

types of EM

transmission EM - samples must be thin/sectioned, electrons pass through the sample to be detected

scanning EM - electrons hit surface of sample produces signals, image detected reveals surface of sample → 3D surface structure

Ab structure

• light chain and heavy chain

• variable region: tips of Y where antigen can bind

• constant regions: everything else

recombination of DNA makes different Abs

B cells

B cells (white blood cell) express one Ab

Ab expressed on surface of B cell recognizes antigen → B cell activated

• differentiates into plasma cells that secrete large amounts of Ab that targetpathogen/antigen of that type, mark for degradation

• clonal expansion - divides rapidly to form more Ab

immunohistochemistry

• use Ab to detect antigens in tissue sample

• Ab usually linked to an enzyme or fluorescent dye

• enzyme substrate is added or dye is activated for detection by light microscopy

immunogold EM

• use Ab to detect antigens

• Ab are linked to gold nanoparticle/bead

• location of Ab indicated by dense black dot in EM

Western Blot

detect changes in

• concentration, modification/substitution/addition to protein, interactions with other proteins

1. protein isolated

2. chem/physical based methods to disrupt membrane

3. detergent solubilize membrane

4. whole cell lysate (nuclear, cytoplasmic) obtained

5. lysate loaded onto PAGE for protein separation

   1. with SDS buffer - denature proteins/linearized and uniformly coat in negative charge

   2. lysate can have dye solution - adds color and has glycerol to make it thicker than SDS buffer

6. protein transfer from gel to membrane (nitrocellulose or unreactive material) that’s more durable via electroelution

7. Minimize nonspecific Ab binding to membrane by incubating membrane with blocking agent like milk or purified proteins in mild detergent

8. Incubated in blocking solution and primary antibody for POI

9. Washes with washing buffer (same as blocking solution/mild detergent remove nonspecifically bound primary Ab) under mild agitation

10. Incubated with secondary Ab (bound to fluorophore) recognizing primary Ab

11. Washes with washing buffer

cytoskeleton function (5)

• cell shape/’structure

• spacial organization

• connect cell to external environment

• movement of molecules within cells

• movement of cells/change cell shape

cytoskeleton filmanent

made of protein subunits that bind to each other noncovalently

actin filaments- 7 nm

intermediate filaments - 10 nm

microtubule - 25 nm

Actin

• actin monomer

• helical

• asymmetrical/polar, 2 ends are different

• diverse structures

• concentrated near cell edge

Microtubules

• tubulin heterodimers

• hollow tubes

• asymmetric/polar

• rigid/straight

• one end = minus end that’s attached to microtubule organizing center (MTOC) near nucleus

Intermediate Filaments

• tetramers

• ropelike structure

• symmetric/nonpolar (ends same)

• strong

• large and heterogeneous group

phospholipid bilayer

hydrophilic/polar head + hydrophobic/nonpolar tails

head = polar group (varies by phospholipid) - phosphate (negative charge) - glycerol

tail = 2 fatty acid chains

form bilayers through hydrophobic effect

cytosolic vs noncytosolic leaflet

a bilayer has 2 leaflets

noncytosolic leaflet - one side extracellular space, one side cytosol

cytosolic leaflet - one side cytosol, one side cytosol, inside = lumen

extracellular environment

• outside of cell

  • water, air

  • protein, carbohydrate, many other molecules

  • cells (same, different types)

cell influences EE and EE influences cell

extracellular matrix (ECM) structure (5)

• 3D molecular network surrounding cells in multicellular organism

• composed predominantly of proteins and carbohydrates

• synthesized and modified by cells

• highly variable components/organization

• contributes to structure/function of cells/tissues

ECM Molecules

polysaccharides, proteins with cov. attached hydrates (glycoproteins, proteoglycans)

i.e collagen, elastin, laminin, fibronectin, glycosaminoglycan, hyaluronic acid

ECM function (10)

• shape and organization of tissues

• mechanical properties of tissues

• physical attachment of cells

• biochemical signaling

important for

• cell migration

• cell differentiation

• wound healing

• development

• diseases including cancer

• tissue enginering and regenerative medicine

cell junctions

interaction with extracellular environment

link outside to inside

cytoskeleton filaments i.e actin - adaptors - transmembrane proteins through noncytosolic leaflet/PM that bind to ECM

Cell-ECM junctions

• transmembrane proteins bind to stuff in ECM

• aligned with actin

• common junctions: actin linked cell matrix, hemidesmosome

Cell-Cell Junction

• contains transmembrane proteins in PM of both cells that bind to each other, can be same or different transmembrane proteins

• transmemb prot - adaptor - cytoskeletal filament

• intermediate filaments align

• common types: desmosome, tight junction, gap junction, adherence junction

cell membrane are diverse, dynamic

diverse: differ in types/amount of lipids and proteins in PM

variation between leaflets, regions, organelles, celltypes

dynamic: lipid/prot move within leaflet: rotation, bending, lateral diffusion

flipping is rare and requires energy

co-translational translocation

prot translation: mRNA read by ribosome, synthesize protein starting at N-terminus

• in N-terminal, many proteins made by CTT have specific sequence called N-terminal ER signal sequence or signal peptide

  • 8+ hydrophobic AAs

  • targets protein for CTT into ER

• Signal Recognition Particle made of RNA and protein binds to signal peptide and ribosome → pause in translation

• SRP binds to SRP receptor/transmembrane protein complex in ER membrane

• SRP binding to SRP receptor all translation to continue + positions ribosome close to translocon/protein complex in membrane with water filled channel

  • when ribosome not bound to translocon, translocon is blocked by part of protein called plug

  • when bound, plug moves so protein synthesized moves through channel

• SRP/SRP receptor released from complex

• ribosome positioned on translocon + protein synthesizing through translocon channel, hydrophobic N-terminal ER signal sequence binds to side of channel near cytosolic side

• signal peptidase enzyme cleaves protein after signal sequence → releases peptide into ER lumen

  • cleaving occurs during or immediately after translation

• N-terminal signal sequence released into membrane by lateral opening of translocon, usually gets degraded

• After translation, ribosome dissociates from translocon

unidirectional, there’s no going from lumen to cytosol

orientation of protein relative to membrane doesn’t change after translation

protein translocation: proteins that go to GA, lysosomes, endosomes, cell surface all need to pass through the

ER membrane

polyribosome

multiple ribosomes bind to mRNA that then gets cotranslated into ER creating the rough ER

water soluble vs transmembrane proteins

completely cross membrane vs embedded into the membrane

same steps as CTT, ribosome positioned on translocon, SRP receptor leaves

signal sequence opens translocon channel, binds to channel as protein threaded through membrane as loop → released into lumen, signal peptidase cuts peptide after signal sequence which is released and rapidly degraded → protein goes to bind and close translocon

signal sequence bound to translocon channel initiates CTT → transfer halted by stop transfer sequence/additional sequence of hydrophobic AAs further in peptide chain → stop transfer sequence released laterally and drifts into plane of lipid membrane → forms membrane spanning segment that anchors protein into membrane

internal signal sequence

in some transmembrane proteins, ISS used to start CTT which continues until stop transfer sequence is reached → 2 hydrophobic sequences released into bilayer where it stays anchored

complex multipass proteins - many hydrophobic regions span bilayer, additional paris of start and stop sequences that reinitiate translocation along the peptide and stops translocation/polypeptide release

• stitched into membrane

single pass transmembrane protein

• N-terminal signal sequence coming out of ribosome associated to translocon, gets translocated

• hydrophobic transmembrane domain/second transmembrane sequence remains in translocon, doesn’t get into lumen → translation resumes

• signal peptidase cuts peptide after NSS

• N-terminus in lumen, C-terminus in cytosol

protease protection assays

experimental method to study proteins and processes associated with membrane and membrane bound organelles

• can be used to study processes like CTT

• determine orientation of membrane proteins in a membrane

protease acts enzymatically on POI → time → protease + degraded POI

“protection” refers to protection of POI or part of POI from protease i.e using a membrane that protects against protease

In vitro PPA

study outside of cellular context

starting sample in tube: POI in membrane bound compartment

• experimental sample: +protease, time to starting sample

  • use SDS-PAGE/WB to detect POI

  • compare btw starting and experimental sample

• control sample for protease activity: +protease +detergent +time

  • disrupt membrane so protease can degrade POI

  • if POI not degraded → protease not working

• control sample for detergent: +detergent +time

  • check if detergent has protease activity

  • membrane disrupted and POI not degraded

ER-Derived Microsomes

study CTT using this in vitro

small membrane bound compartments derived from ER

rough ER with ribosomes on cytosolic side → disrupts → smaller compartments called microsomes

• rough and smooth microsomes (±ribosomes)

• can be separated by different densities

  • mini-ERs, support functions like CTT

study CTT in microsomes with PPA

starting sample: rough microsome + components for prot translation: mRNA< amino-acyl tRNAs, GTP, translation factors

+time = protein translation occurs, protein located in microsome following CTT

+protease + time = protease acts

• use SDS-PAGE or WB to detect POI

  • if POI in microsome → not degraded

additional samples as controls or experimental conditions

orientation of internal ER signal sequences (ISS)

N-term - (+)ISS(-) - C-term → N-terminal in cytosol, C-terminal in ER lumen

N-term - (-)ISS(+) - C-term → C-terminal in cytosol, N-terminal in ER lumen

aka positive is inside cytosol

ISS vs TMD

internal signal sequence - anchor and targeting sequence for CTT, hydrophobic AAs

transmembrane domain - anchor in CTT, hydrophobic AAs

protein processing in ER (3)

3 major processes

1. folding

2. disulfide bond formation

3. glycosulation

folding (protein processing ER)

• can occur cotranslationally as soon as it exits ribosome

• molecular chaperones are proteins that help others fold properly

  • bind to unfolded protein, incorrectly folded protein, and unassembled component of protein complex

  • chaperone binding/unbinding often coupled with ATP hydrolysis

  • in ER: BIP, calreticulin, calnexin

disulfide bonds (protein processing ER)

• covalent bonds between two sulfurs on 2 cysteine AA sidechains

• formed due to oxidizing environment

  • two -SH sticking out, remove Hs, S-S

• protein disulfide isomerase - protein in ER that helps formation of correct disulfide bonds

• form between 2 cysteines on same protein or 2 different proteins

glycosylation (protein processing ER)

• most proteins synthesized at ER have covalently attached carbohydrate

• carbohydrate attached as single unit to asparagine AA in protein

  • asp/N - X - S or T

• linkage catalyzed by enzyme in ER membrane

• N-linked glycosylation helps proteins fold properly

N-linked glycosylation helps proteins fold properly

carbohydrate attached to Asn → two glucoses of three removed by enzymes → monoglucose carb bound by chaperone calreticulin/calnexin to help protein fold → protein separated from chaperone by removal of final glucose by glucosidase

if protein misfolded, it will bind to glucosyl transferase (adds back glucose), so then chaperones can rebind and refold

proper folded protein - no glucoses on it

ubiquitination

covalently attaching ubiquitin - small protein of 76 AAs to other proteins

attachment is between C-terminal of ubiquitin and sidechain of lysine AA or N-terminal of target protein

• catalyzed by ubiquitin ligase (also known as E3 enzymes, E1 and E2 also involved)

• removed by deubiquitinating enzymes

single ubiquitin attachment = monoubiquitination

polyubiquitination

since ubiquitin has lysine residues, one ubiquitin can be attached to another ubiquitin to form a chain

use K6, 11, 27, 29, 33, 48, 63 or N-terminus (M1)

• linear or branched

added by ubiquitin ligase, removed by deubiquitinating enzymes

ubiquitination alters target protein structure and function

creates binding sites for other proteins with specific ubiquitin binding domains

linkage types determines outcome of ubiquitin change

has many roles in

• regulation of transcription

• DNA repair

• nuclear transport

• protein degradation

• cell death, signaling, division

ER-Associated Degradation (ERAD)

• process of degradation of misfolded proteins in ER

• removes nonfunctional proteins from ER

• proteins in lumen and ER membrane that aren’t able to fold correctly are substrates for degradation

ERAD process (4 steps)

1. recognition of misfolded protein

proteins translated by CTT, start unfolded in lumen and fold

molecular chaperones help fold + recognize if protein unable to fold within a reasonable timeframe

2. retrotranslocation

Transport of recognized misfolded protein across lipid bilayer into cytosol through retrotranslocon.

3. polyubiquitination

Unfolded protein is polyubiquitinated

4. proteasomal degradation

Polyub chain targets protein for degradation by proteasome

Proteasome is large protein complex present in cytosol, protein enters middle of proteasome that has multiple proteases that completely degrade the protein

Ubiquitin proteins are removed before degradation so they can be reused/recycled again

guanine nucleotide-binding protein (g-proteins)

• bind guanine nucleotides

• bind GDP or GTP (diphosphate vs triphosphate)

• GTPases that catalyze hydrolysis of GTP→GDP

  • enzymatic activity/GTPase activity

GTPase activity

G-proteins have enzymatic activity: GTP → GDP

G-proteins must release GDP molecule → empty G-prot → rebind GTP if conc. high enough

• in cells, GTP concentration is typically way higher than GDP conc

GTP bound form = active conformation

GDP bound form + empty form = inactive conformation

GTPase Activating Proteins (GAP)

increase GTPase activity

stimulate hydrolysis of GTP → GDP by G-prot

favor inactive state

guanine nucleotide exchange factors (GEFs)

stimulate GDP release by G-prot → empty G-prot

favor active state

G-Proteins act like molecular switch

• either on or off

• can be switched quickly between two states

• key for temporal and spatial regulation of cellular processes

vesicle transport

• exchange of molecules occurs through vesicles: small membrane compartments, between endomembrane system: ER, GA, nuclear envelope, endosomes, lysosomes, PM, biomolecules

• vesicles are formed at one membrane and fuse at a different membrane

• aka membrane trafficking, vesicle trafficking

topology is maintained during transport

orientation of molecules relative to cytosolic/noncytosolic side of membrane is maintained

• luminal in ER → as it moves, it will stay inside lumen/noncytosolic

• transmembrane in ER → as it moves, N/C terminus will stay oriented i.e N-terminus noncyt/C-terminus cyt

vesicles formed at particular cellular membranes have specific target membranes (3)

1. secretory

2. endocytic

3. retrieval

secretory pathway

two types: ER → PM, ER → lysosome

ER → PM

1. ER → GA (early compartments → late comparments)

2. exocytosis: late GA → PM → release molecules into extracellular space

3. secretory vesicle = specialized vesicle that delivers specialized cargo molecules to extracellular space

ER → lysosome

1. ER → GA

2. GA → early or late endosomes

3. early endosome → late endosome

4. late endosome → lysosome

endocytic pathway

molecules located in extracellular space or at PM to move into cell towards lysosomes

PM → lysosome

1. PM → early endosome → late endosome → lysosome

retrieval pathways

in order to function, need molecules located in specific place

molecules can be transported to other compartments in a process that is part of their function or nonspecific process

used for molecules that need to be returned from one location to a different location in order to function properly

1. GA → ER

2. late GA → early GA

3. early or late endosomes → late GA

4. early endosome → PM directly or early endosome → recycling endosome → PM

5. secretory vesicle → late GA

vesicle budding

vesicle budding from donor membrane - process of curving/bending membrane into cytosol to form small separate membrane bound compartment

1. can be initiated with transmembrane cargo proteins with specific signal sequence

2. adaptor protein complex binds to signal sequence in cargo protein, link cargo protein with coat protein complexes

3. self assembly of coat protein complex leads to membrane curvature/bending and cargo clustering within forming vesicle bud

coat recruitment GTPase

regulate association of coat proteins, specifically with donor membrane

coat recruitment GTPases located in cytosol in inactive or GDP bound state

1. nucleotide exchange and activation of coat recruitment GTPase is stimulated by GEF in membrane

2. coat recruitment GTPases swap GDP for GTP → conformational change causes hydrophobic part of protein to become exposed → associates with lipid bilayer of donor membrane

3. GTP bound coat recruitment GTPase binds to coat protein complex bringing it close to the membrane

vesicle scission

pinching off of vesicle occurs in small region still connecting vesicle to donor called neck

vesicle targeting

association of vesicle with a specific membrane and the movement of vesicle close to target membrane

transmembrane proteins in vesicle membrane and target membrane mediate targeting

1. vesicle tethering - first association of vesicle with target membrane is through long range association

2. vesicle docking brings vesicle and target membrane close together

vesicle tethering

involves Rabs - GTPases that can be in GDP bound/inactive or GTP bound /active state, have a covalently attached lipid group, GDP dissociation inhibitors - shield hydrophobic lipid group and keep Rab soluble in cytosol when Rab bound to GDP

1. Rab GEF promotes nucleotide exchange GDP → GTP, regulates association of Rab with a specific membrane

2. In GTP bound state - Rab unbinds GDI and exposed lipid embeds in a membrane i.e vesicle, target, or vesicle & target membrane

3. Rab GTP in membrane can bind to 1+ Rab effector proteins - proteins that bind Rab only in GTP state

• some Rab effector proteins are membrane bound proteins that act as tethering proteins to link vesicle and target membranes together

vesicle docking

close association of vesicle and target membrane

accomplished by transmembrane proteins called SNAREs in both vesicle and target membrane

associated with vesicle = v-SNAREs

• consist of single protein with one helical domain

associated with target membrane = t-SNAREs

• consist of 3 different proteins each with one helical domain

4 helical domains of SNAREs interact to form four-helix bundle that acts like zipper to bring membranes very close together

association of v-SNAREs and t-SNAREs is very specific

vesicle fusion

after vesicle targeting brings vesicle very close to target membrane, vesicle fusion can occur

association of SNAREs involved in membrane fusion too

1. formation and zipping up of four helix bundle excludes water from space between vesicle and target membranes

2. very close association of two membranes allows for fusion of cytosolic leaflets of membranes

3. fusion of noncytosolic leaflets occurs to complete fusion

4. following fusion, lipids from vesicle are part of donor membrane, lumen of vesicle merged with lumen of target membrane

vesicle process + vesicle uncoating + vesicle movement

1. vesicle budding

2. vesicle scission

3. vesicle…

   1. UNCOATING: coat proteins come off of vesicle, happens at different times/ways for different coat proteins

   2. MOVEMENT: vesicle gets moved by motor protein along cytoskeletal filament (microtubule)

4. vesicle targeting

   1. tethering

   2. docking

5. vesicle fusion

vesicle coats

COPI, clathrin

outer layer = coat proteins

• coat proteins, coat recruitment GTPases

inner layer = adaptor proteins

• bind to transmembrane cargo proteins

COPI and clathrin look different because coat proteins have different structures

resetting system for vesicle transport

in order to recycle components, need to be reset and get back to right place

• coat recruitment GTPases hydrolyze GTP, release membrane, go bind to another donor membrane

• coat proteins disassemble and can be recruited somewhere else

• Rabs hydrolyze GTP and unbind effectors

• v-SNAREs and t-SNAREs separated (need energy) by a protein

  • TMDs on each and 4 helix bundle

why is there specificity of coat protein based on vesicle formed at a membrane

this is about vesicle formation: adaptor → coat recruitment GTPase → GEF

location of GEFs for coat recruitment GTPases is the key

adaptor proteins are binding to at membrane

NOT coat protein

why would a vesicle fuse with different target membranes

vesicle targeting: tethering/docking

based on Rabs (and Rab GEFs) and SNAREs (v-SNAREs)

NOT coat protein

golgi apparatus

• multiple flattened compartments (cisternae) arranged in stacks

• vesicles form/fuse with cisternae

• cis/trans side

  • cis golgi network - cis golgi - medial golgi - trans golgi - trans golgi network

• proteins made by CTT in ER are transported ER → cis golgi → trans golgi

  • protein gets processed moving through golgi

protein gets processed in golgi

sequential processing as protein moves cis → trans

protein processing carried out by enzymes localized to specific GA compartments

• N-linked glycosylation

• addition/modification of O-linked glycosylation: covalent attachment of carbohydrate to oxygen atom of Ser/Threo sidechain

• sulfation + sulfate

• phophorylation + phosphate

• lipidation + lipid

• proteolysis - cut protein in specific places

golgi vesicle transport destinations (4)

1. vesicles at cis-golgi fuse with ER

2. intra-golgi - can fuse with adjacent compartment of GA

3. trans-golgi fuse with endosomes

4. trans-golgi fuse with PM

cis golgi → ER

• COP1 coat protein complex

• cargo protein require specific signal sequence for transport