biology 2020 section 3 (3pp)(1)

Section 3Cytoplasmic

Membrane Systems

Chapter 8Cytoplasmic Membrane

Systems: Structure, Function, and Membrane Trafficking

Required Readingpages 270-305, 308-317

( S e c . 8 . 1 - 8 . 6 , 8 . 8 , 8 . 9 )

P L U SC h a p t e r 1 2 , p a g e s 5 4 1 - 5 4 2

( S e c . 1 2 . 7 )

Clicker Quiz #4

Will be on...

From the beginning of Section 3to (but not including) the slide

that says Membrane Lipid Synthesis

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Eukaryotic Cell Internal Structure

Goal of this section

To answer how:

1. The cell synthesizes components of the membranes

2. The cell ensures that the correct molecules arrives at the correct membrane, or inside the appropriate organelle

Compartments are not independent - they form an integrated functional unit

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VAIOTypewritten Text- all membranes are constantly in flux- very dynamic

VAIOTypewritten Text- compartments all depend on one another- compartments all work together- membranes take on different properties when they reach a certain organelle

Endomembrane System

Endoplasmic reticulum Golgi complex Endosomes Lysosomes

Endoplasmic Reticulum

Rough endoplasmic reticulum (RER)

Smooth endoplasmic reticulum (SER)

lumenor cisterna

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VAIOTypewritten Text- Rough ER (pancake shaped): Ribosomes (rough part) are placed on the ER. - The ribosomes inject proteins inside the ER

VAIOTypewritten Text- compartment inside the RER is the lumen or cisterna of the ER- Smooth ER (tubular shape) : the inside of the ER is never in contact with the cytoplasm. - environment inside is different from the environment in the cytoplasm- the mitochondria and chloroplasts are outside of the innermembrane system.

SER Functions

Synthesis of steroid hormone

Detoxification Oxygenases

Cytochrome P450

Glucose release

Sequestering Ca++

Rough Endoplasmic Reticulum

Protein secretion

Lumen proteins

Membrane proteins

MembraneProtein

Synthesis

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VAIOTypewritten Text- proportions hange in different tissues- detoxification in liver : oxidize- cytochrome P450 (oxygenase) makes hydrophobic molecules hydrophillic- Ca++ is important for cell signalling

VAIOTypewritten Text

VAIOTypewritten Text- lumen is inside

MethodologyPages 267-273(section 8.2)

CellFractionation

Microsomes

CellFractionation

Microsomes

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VAIOTypewritten Text- should read sec. 18.4 - labratory tecniques

VAIOTypewritten Text- cell fractionation : two approaches1. use a microscope to look at a single cell itself2. take a whole bunch of cells, mash them up, seperate comonents and analyze seperately- take cells and put them in a tube with buffer (liquid thats isotonic), use a plunger thing fits inside tube with the cells and buffer. smash cells approx 100. times on ice.- take homogonate and spin it in a centrifuge at a med. speed. Have a seperation of the different compartments of the cell. Mito, nuclei go to the bottom of the tube- transfer supernatant to a new tube, spin at much higher speeds using an ultracentrifuge (50,000 G for 2 hr)- seperate what is left (postmicrosomal supernatant and microsomes. In the microsomes, you rupture the membranes of the cell components, most of these membranes are ER)- possible to see that proteins are made in two seperate places in cells :- cytoplasm and ER

Secretion Pathway

Cell fractionation Autoradiography

Pulse chase

Autoradiogramof

pulse chaseexperiment

We can figure out where proteins travel by doingpulse-chase experiments in combination withcell fractionation or autoradiography

Thats great.But how do we figure out how proteins actually get secreted?

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VAIOTypewritten Text- can be used with the previous technique- pulse chase experiment: to figure out how cells get secreted and which places they pass by to go out of the cell- they gave the cells media that contained radioactive amino acids (cystines), because sulfur in cystines can be radioactive (have a radioactive isotope). The cystine then gets incorporated into the newly made proteins. the protines that have a cystine will have the radioactive a.a- red dots are newly made proteins that have been incoroporated in golgi complex that are labelled with radioactive isotopes. (pulse when they give radioactive to cells) (chase when they give normal cells) - all the other proteins that are made after that wont have radioactive. The newly made proteins : 3 min still in ER. 20 min: go to golgi- found that all protiens go to golgi before they go out of the cell

VAIOTypewritten Text- once you do pulse chase, you can do 1. cell fractionation or 2. autoradiography (you look at the individual impact cells. - printed film using radiographic emultion- take fixed cells in their plates and cover the plates with liquid emultion that contains the silver grains. When exposed to light or radiation, they become black. You can see that some parts of the cells will have the silver grains that turned black .... it shows where the proteins have travelled to. ER > GOLGI > ENDOSOMES > proteins leave


VAIOTypewritten Text- used yeast cells. With the yeast cells, the were able to make mutations in certain proteins by treating the cells with certain chemicals (mutagens). They looked at different strains of yeast cells that had mutations, some ofthem had problems with protein secretion. They screened mutations to see which proteins had problems with protein secretion. Able to isolate secretion mutants (SEC MUTANTS ). Screened by doing the pulse chase experiment and autoradiography on thousands of strains to find the mutants that had problems with the secretion pathway. Called SEC mutants. They were classified in 5 different classes

Yeast SEC Mutants C

E

D

BA

So, an experiment would go like this.....

Grow temperature sensitive mutant yeast and non-mutant control at normal temperature (room temp)

Pulse chase

Autoradiography

Raise temperature to activate mutation (about 36oC)

Observe under microscope

Endomembrane System

Endoplasmic reticulum Golgi complex Endosomes Lysosomes Plasma membrane

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VAIOTypewritten Text- CLASS A: proteins didn't leave the cytoplasm, didn't even enter ER. Found that The gene that's mutated is probably a gene that encodes the proteins that's necesarry for the newly made protein (nascent protein) to enter the ER. accumulate in the cytosol-CLASS B: Nasecnt proteins aren't getting in ER, they accumulate in RER, but can never leave. Probably whatever protiens that are important for nascent to go to golgi aren't functioning.- CLASS C: proteins accumlate from ER > golgi transport vesicles. - CLASS D: accumulation in golgi-Class E: accumulation in secretory vesicles... - had to select for mutants that they can still grow, like yeast. If mutations are always active, the cells would just die. They made mutants without having cells die because the mutants are heat-sensitive mutations. They are called that because the mutations that they introduced make some of the proteins more fragile, but still works at normal growth temperature. When you raise the temperature, the protien starts to unravel and denature. That's why the mutation is heat sensitive.

VAIOTypewritten Text1. need a control, always grew mutant proteins with a control cell (no mutation) at room temp2. raise temp to activate. For yeast, 36 degrees is warm3. pulse chase4. autoradiography5. observe- probably did a thousand times + - able to isolate the gene






2 biosynthetic (secretory) pathways

Constitutive secretory Regulated secretory

(storing in secretory granules)

Endocytic pathway

vesicle transport

lumen = inside of vesicle = outside of cell

Summary

The endomembrane system comprises the smooth ER, rough ER, golgi, lysosomes, endosomes

Several techniques and systems have been developed to study vesicle/protein transport: examples: pulse-chase (analysis by membrane fractionation and autoradiography), yeast mutants

2 biosynthetic pathways: constitutive secretory, regulated secretory

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VAIOTypewritten Text- 2 biosynthetic pathways found:1. constitutive 2. regulated- both are the same all the way till the vesicles that pass the golgi. The vesicles are regulated differently1. vesicles go automatically to the outside of the cell. Not regulated2. vesicles are called secretory granules, they accumulate right underneath the surface, when the proper signal is given, they then expell their content outside (ex. synapses)3. endocytic pathway: cell will take things inside. The way the cell regulates what's on its surface

VAIOTypewritten Text- the lumen of any compartment(ER, golgi, lysosomes) is always seperate from the cytoplasm, even when you have a vesicle forming that takes in materials, the green is never in contact with the white. The receptors always maintain the same orientation.- membrane that points towards cytoplasm iis always going to be the same- membrane that faces towards lumen is always going to be the same membrane- when a vesicle fuses to the plasma membrane and expels its content, its the luminal side of the vesicle that gets expelled. Membrane inside the lumen eventually becomes the outside leaflet of the plasma membrane.


VAIOTypewritten Text-


There are many ways for proteinsto move between cellular compartments

We know that vesicles shuttle material between

compartments...

but how do proteins get incorporated in these?

from Lodish et al., MCB

Some ribosomes are attached to the ER and some are not

Ribosome

newprotein

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VAIOTypewritten Text- different ways for proteins to move around in the cell



VAIOTypewritten Text- proteins getting shuttled are part of the membrane of the vesicle


VAIOTypewritten Text-ribosomes translate information from mRNA into protein- makes new proteins that are incorporated inside the lumen of the ER

A very important doodle!!!

from Gnter Blobel Nobel Lecture, December 8, 1999FIRST PUBLISHED IN 1971

The Signal Hypothesis1. Ribosomes are all the same2. Its a signal on the protein that tells it where to go

Blobel in 2008(Wikipedia)

Ribosomes are all the SAME(no special type for proteins destined to cytosol, nucleus, ER...)

ER lumen

Freeribosomes

Membranebound

ribosomes

An important experiment

Protein made in vitro

Protein made in vivo

Blobel and Sabatini deconstructed the cell and isolated the parts necessary for in vitro translation. This was key to proving their hypothesis.

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VAIOTypewritten Text- subunit pool of ribosomes... they clamp onto the mRNA

VAIOTypewritten Text- can have alot of ribosomes clamping onto the same mrna- called polyribosomes

VAIOTypewritten Text- had to deconstruct the cell, take parts that are necessary for invitro translation- mRNA, ribosomes, tRNA- made protein invitro, bigger than ones made in vivo..... even though they're the same protein because protiens made in vitro had a single sequence. In vivo the single seqcence gets cleaved offf. The difference in size is the single sequence


The signal peptide is the postal code of the protein

(sequences FYI)

CotranslationalTranslocationMechanism

Signal Sequence Hypothesis

GTP Signal peptidase

BiP Calnexin

HOLD!

See Fig 8.12

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VAIOTypewritten Text- some for import and export.... etc.- higlighted in yellow are hydrophobic amino acids

VAIOTypewritten Text- ribosome clamps onto mRNA, tRna comes and add amino acids as mrna is being read- signal seqcence gets recognized by SR particle (composed of 6 protein)- SR particle recognizes amino acids and clamps onto the amino acid-SRP receptor faces towards the cytosol and clamps onto the SRP- GTP used as a molecular fuse (timer). When SRP becomes active, it binds to GTP. Proteins that bind to GTP are called GTP ases- GTP then gets hydrolied to GDP and this allows the SRP particle to leave- nascent proteins bind to chaperones to allow the protein to fold properly without any interference from the other protiens. Signal peptidase cuts of signal peptide

Protein synthesis on the rough ER Includes: secreted, lysosomal, membrane bound, reside in

ER or golgi

1. translation starts on a free ribosome

2. signal peptide holds translation and SRP binds to signal pep.

3. SRP docks the ribosome to a SRP receptor (both bind GTP)

4. SRP release and move to translocon

5. translocon plug is removed by elongating peptide, signal peptide is cleaved by a signal peptidase

6. Chaperones bind to elongating peptide and ensure proper folding

Problem:

How do proteins get integrated into the

membrane?

The signal-peptide does not have to be at the beginning!

from Alberts et al., MBC

Start-transfer sequence

sequence

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VAIOTypewritten Text4. SRP moves to transolocon and gets released. Once get released, translation can continue

VAIOTypewritten Text- differenceis that signal peptide is internal and doesn't get cleaved off- internal start-transfer sequnce is hydrophobic, It's borderd by negative and postive charged amino acids- positivley charged a.a are in the cytosol side





From http://bcs.whfreeman.com/lodish6e/go to: animations > select Chap 13 > select Synthesis of...

The signal sequence can snap into the translocon in the other direction depending on how the protein is encoded(this orients the protein the correct way)


Membrane proteins can have many many transmembrane domains...

3+

-

in

out

...But all proteins use the same basic mechanism to get in

(eg. a voltage gated sodium channel)

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Double-pass transmembrane protein


STOP-transferSTART-transfer

Matureprotein

NH2 COOH+ - + -+- +-

+ + + +

- - - -

NH2 COOH

starttransfer

stoptransfer

starttransfer

stoptransfer

cytosol

ER lumen

MembraneLipid

Synthesis

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VAIOTypewritten Text- two types of internal signals that don't get cleaved off. They both become the transmembrane domains of the protein- can have multiple start transfer and stop transfer

VAIOTypewritten Text- has four transmembrane domains- stop transfer is a point where there's a signal where the ribosome is told to stop to thread the protein through

EXOPLASMICLEAFLET

EXOPLASMIC FACE

CYTOSOLIC FACE

CYTOSOLICLEAFLET

OR LUMEN

Membrane Synthesis and Asymmetry

MembraneSynthesis andAsymmetry

Leaflet

Cytosolic

Exoplasmic

Clicker Quiz #5

Will be on...

From Membrane Lipid Synthesisto (but not including) the slide on

Targeting and Vesicle Fusion

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VAIOTypewritten Text- cytosolic leaflet faces the cytosol-exoplasmic leaflet faces either the lumen ofa vesicle or the outside of a cell

VAIOTypewritten Text- keep same orientation throughout the biosynthetic pathway (until reaches the surface of the cell)- membranes maintaing symmetry

Membrane Lipid Synthesis

from LodishLUMEN

CYTOSOL

1. Phosphoipids are inserted intopre-existing membranes (in the ER)2. Phospholipid synthesis occurson the cytosolic face3. Flippases are needed fortransfer to exoplasmic face

Modification by enzymes

Vesicle formation

Phospholipidtransferproteins

Why is the lipid composition of organelles different?

Summary membranes arise from pre-existing membranes

(components are added in the ER)

Asymmetry is maintained Synth of phospholipids occurs on the cytosolic face and

are transfered to the exoplasmic face via a flippase

Lipids are then carried from the ER to the Golgi via transport vesicles

The action of enzymes (in the lumen), differential inclusion of lipids in vesicles and phospholipid transfer proteins produce membranes with different compositions in different organelles

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VAIOTypewritten Text- there's a cytosolic side and a lumen side. The ER is where the lipids get made on the cytosolic side. The enzymes have their active sites pointing towards cytosol where action happens. Once the phosopholipids are made on cytosolic side, they need flippase to flip to exoplasmic side. - lipids are made in pre-existing membranes

VAIOTypewritten Text- intitially lipid membranes are similira. The structures can be transformed because there are vesicles (lysosomes) that don't have the same structure of certain organelles. - need mechanism to change structure after lipids are made : 1) modification by enzymes change lipids after they are made. 2) Preferential inclusion or exclusion of certain lipids into vesicles and carry them to another organelle. 3) Phospholipid transfer proteins, where enymes pluck certain lipids from one membrane andput it inside another membrane.


VAIOTypewritten Text- before the lipids go to the plasma membrane, they go through the golgi

Post-translational Protein Modifications

Disulfide bond formation

Folding

Proteolytic cleavage

Multimer assembly

Glycosylation

N-linked

O-linked

Glycosylation

Glycosyltransferase

Nucleotide sugarCarbohydrate chain

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VAIOTypewritten Text- disulfide bond formation: bonds formed between cystines- folding: folding of the protein occurs during translation, but mostly afterwards- proteolytic cleavag: sometimes smaller proteins are made as larger protein. cutting up enzymes- multimer: some proteins can have more than one subunits- glycosylation:


VAIOTypewritten Text- n or o linked. O-linked is more common.- n-linked is called n because they are linked on the asparagine. First sugar monomer side chain attached to asparagine is N-Acetyl.....

VAIOTypewritten Text- when sugars are being built, oligosaccharides (10-30 monomers) made from precursors that are attached to the nucleotide carrier. This carrier allows the monomer to get recognized by the enzyme. The enzyme that builds the sugar trees are called glycosyltransferase (transfers sugar to new growing tree)

N-linked Glycosylation

DolicholLUMEN

CYTOSOL

Oligosaccharide-protein transferase

ProteinQuality Control

Qualitycontrol

Monitoring EnzymeUGGT

Calnexinor

Calreticulin

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VAIOTypewritten Text1. N-linked occurs at the endoplasmic reticulum. the sugar residues don't get added immediately to the protein. We need a holder (special lipid called dolichol). initially the glycosolations add the first few monomers to the dolichol initally. The initial carbs (oligosaccharides) point towards the cytosol. 2. special kind of flippase that flips the dolichol towards the inside of the lumen3. oligosaccharide protein transferase enzyme, The oligosaccharide continues to grow until its ready to be plucked from dolichol and added to growing peptide. - consensus sequnce for addition of n-linked is asparagine, and any a.a and either a cyrine or thyrine, than the enzyme (oligosaccharide-protein...) plucks one of the sugar trees being mae and adds it to protein. Protein can have several oligosaccharides


VAIOTypewritten TextN-linked glycosylation summary- occurs at the ER. Entire oligosaccharide is made before being added to nascent peptide.- special lipid, dolichol, acts as a holder for the growing carb branches- glycosyltransferases add monomers to branches- carb faces cytosol during early synthesis. Then flipped to lumen- oligosaccharide protein transferase transers oligosaccharide to peptide

VAIOTypewritten Text- protein quality control: once proteins are made, they don't behave how we want them to

VAIOTypewritten Text- when protein is made, chaperones are bounnd to the hydrophobic sites.




VAIOTypewritten Text- unfolded protein, glucosidase (enzymes that cleave glucose) are recognized by an enxyme called calnexin (membrane bound) or calreticulin (both chaperones) they recognize oligosaccharide by binding to the glucose and gives protein a chance to fold properly. After some time the protein leaves. If protein is properly folded it will be able to exit the ER. If not correctly folded, hydrophobic a.a. point towards outside (not properly orientated have to point inside). UGGT recognizes hydrophobic a.a that aren't oreintated properly in protein. Protein not properly folded is recognized by UGGT protien or other enzyme (it sends the unproper protein back to translocon, sent to cytosol (reverse translocation). Protein that isnt properly folded bumps into UGGT. another glucose is added to another oligosaccahrdie, recognize by calnexin, another chance to fold properly. Then detach and goes through process again. If not able to fold properly, it'll be sent towards the cytoplasm

Proteasome Sec. 12.7Pg. 541-542

E1, E2, Ubiquitin ligase (E3)

ATP

Unfolded Protein

Response

Nucleus

ER Membrane

ER lumen

cytosol

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VAIOTypewritten Text- two main players in regards to proteins that are sent back to cytosol through translocation. Proteasome is the protein destroyer. It chews up proteins, these proteins first have to be tagged. The tags that are placed on proteins that are meant to be destroyed. They are smal protines (8.5 kDa) added to other proteins to mark them for destruction. Called ubiquitin


VAIOTypewritten Text- 3 enzymes that recognize proteins that are supposed to be destroyed. Necessary for adding ubiqutiin. E1 AND E2 recongize the proteins, recognizes hydrophobic poining to the outside. E3 called ubiqutin ligase (something that ties. The E3 will work with E1 and E2 to add ubiqutin on the protein.)- can have several that are added back to back- E1 & E2 bound to hydrophobic side of protein. E3 recognizes and adds ubiquitin- proteolysis occurs in Beta



VAIOTypewritten Text- occurs when there is stress, like heat (causes protein to unfold/denature). Cell evovled this mechanism to keep its proteins folded properly1. alot of unfolded protein, transmembrane protein sensors sense proteins that are being misfolded (when there is no stress, the chaperones bind to sensors weakly.When there are a lot of misfolded, they want to bind more to hydrophobic sites. Chaperones on sensors act as inhibitors) Gets activated by forming pole dymers. Two kinds of sensors: inactive sensors and activated sensors. One sensor acts as a kinase. 2. translation factor when phosphorolated binds to the protein 3. makes more mRNA for proteins like chaperones and other proteins capable of removing stress.

If nothing works....

Apoptosis(programmed

cell death)

Summary In the protein Quality Control process, the calnexin (or

calreticulin) chaperone uses glucose as a marker for folding while the GT protein detects misfolded proteins and adds a glucose if another round of folding is needed

If the misfolded state persists, the protein is sent to the cytosol by reverse translocation (through the translocon) where the protein is polyubiquitinated and degraded by the proteasome.

If proteins cant be sent to the cytosol quickly enough, the Unfolded Protein Response comes into play. Gene expression of chaperones, coat proteins and QC proteins is increased while overall translation is decreased.

If this does not work still, the cell commits suicide (apoptosis).

Endoplasmicreticulum

Golgi Intermediate

Compartment(ERGIC)

Endoplasmicreticulum

Golgi Intermediate

Compartment(ERGIC)

Vesiculartubular cluster(VTCs)

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VAIOTypewritten Text- for well folded proteins- transported to another organelle called the golgi apparatus. After protein translation, post-translation modification occurs inside the golgi.- Transition stage between ER and golgi- vesicles are formed and are coded with proteins- ERGIC area between the ER and golgi (area). There are vesicles in ERGIC called vesicular tubular clusters VTC - VTC become the golgi and they bud from the rough ER and fuse into the VTC to become the golgi

Camillo Golgi

Invented silver-based histological stains

Allowed the visualization of many new cellular structures

1906 Nobel Prize in Medicine for the study of structures of the nervous system

Golgi Complex Cisternae

Golgi Complex

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VAIOTypewritten Text- developed silver based stains because silver binds to proteins.

VAIOTypewritten Text- cis-Golgi network is where things enter. They exit at the trans-golgi. - as there are more and more mebranes full of proteins to the VTC, it fills in the holes in the golgi so that you no longer have tubules. The stack of tubules is being built underneath and move. The ones being made are the new ones- cis cisternae have a lumen in the middle


Stack ofelectron

micrographs

3-D tomographic reconstruction of a Golgi apparatus

The seven cisternae thatcomprise the Golgi

CISC1, light blue;C2, pink;C3, cherry red;C4, green;C5, dark blue;C6, gold;C7, bright redTRANS

The ER (yellow) forms a single continuous compartment within the modeled region, traversing the Golgi ribbon at multiple points, extending in opposite directions beyond the cis-most and trans-most cisternae

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N-linked Glycosylation in the Golgi

Glycosyltransferases

(EXAMPLE ONLY, STRUCTURE AND ENZYME NAMES FYI)

CIS TRANS

AnterogradeRetrograde

Evidence for cisternal maturation

New proteins being secreted are never seen in vesicles, they remain in the cisternae.

The only vesicles seen travel in a retrograde direction (from trans to cis) and contain Golgi-resident enzymes.

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VAIOTypewritten Text1. enzymes placed in different stacks in the cisterna. In cis golgi you have the alpha, trims sugarsreason you have a lot of sacs is for modification to happen



VAIOTypewritten Text- probelm with anterograde: didn't see nascent proteins travelling in the anterograde direction. You should see the nascent proteins in the vesicles. Instead the membranes of the trans-golgi go backwards in the retrograde direction.The newly made proteins remained in the cisternal sacs. - cisternal maturation model: new proteins that are being secreted are never seen in vesicles while in the golgi because they remain in the cisterna. The only vesicles seen that travel are travelling in the retrograde direction. This is because

VAIOTypewritten Text- cisternal maturation model: new proteins that are being secreted are never seen in vesicles while in the golgi because they remain in the cisterna. The only vesicles seen that travel are travelling in the retrograde direction. This is because.- a

From http://bcs.whfreeman.com/lodish6e/go to: animations > select Chap 8 > select Protein secretion

Animation:Protein transport through the Golgi apparatus

Vesicle Transport

Role of protein coats:

1. Mechanical device

2. Selects componentsto be carried

COPII-coated forward from ER to ERGIC and Golgi

COPI-coated retrograde from ERGIC and Golgi to the ER and from trans Golgi to cis

Clathrin-coated TGN to endosomes and lysosomes; plasma membrane to cytoplasmic compartments

Coated Vesicles

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VAIOTypewritten Text- resident golgi eznymes are what stays in the golgi. They are brought back to the newly made golgi

VAIOTypewritten Text- vesicles are made on the ER and needed to be coated by special proteins called protein coats. they act as 1. a mecahincal device: by binding to the membrane, it curves the membrane to make the vesicle that will bud off the ER. 2. selects components to be carried, selects proteins that are supposed to go to the next compartment.

VAIOTypewritten Text- three types of protein coats:1. COP2: used for making movement go forward from ER to ERGIC to Golgi2. COP1 : for retrograde movement- everything that happens after the golgi, is mediated by clathrin coat:

Coated Vesicles

COPI

COPII

Clathrin

COPII-coated Vesicle Assembly

Sec24p

Disassembly: Hydrolysis of GTP on Sar1

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VAIOTypewritten Text- need coat proteins to form a protein coat. Also need a nucleating protein that initiates the formation of the coat. For COP2 it's called Sar1, which uses GTP as a timer like the SRP receptor. Whenever sar1 binds to GTP it's able to bind to COP1 proteins. The COP proteins stick to the cargo receptors and form the coat on the cytosolic side. Golgi enzymes are newly made transmembrane proteins that need to be brought to the golgi. Cargo binds to cargo receptors. The coat proteins are going to bind to the part of the receptor that sticks out on the cytosolic side. The red nascent proteins need to be secreted and go to the golgi, The coat then has to fall off. Once the vesicle has budded off, you don't need the coat anymore. Happens when GTP is hydrolized to GDP. Vesicle then gets transported to the next compartment using motor proteins.Sec24p is a coat protein found in mutants (gives name to protein). There are several diff kinds of cop2 coats, that select for different content. All called cop2 beause they go from er to golgi. Once the vesicle has been made and is budded off, the coat proteins fall off, and other proteins replace the coat proteins

COPI Coated Vesicles

Retrograde transportTrans to cis direction in Golgi

ERGIC and Golgi to RER

ARF1

RetrievalSignalsKDEL

lys-asp-glu-leu(soluble prot.)

Binds to KDEL receptor

KKXXlyslys-X-X

(membrane prot.)Binds to COPI

If this protein gets accidentally sent to the Golgi, it will be transported back to the ER

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VAIOTypewritten Text- mechanism to bring back proteins is based on cop1 (trans to cis direction)- same as cop2 with the exception that the nucleating protein that binds the coat is ARF1 instead of sar1

VAIOTypewritten Text- formation of the coat is the same, but the proteins that are ER residents have a retrival signal on them which is alwalys the same (KDEL). The chaperones have the KDEL proteins. -KDEL proteins all bind to KDEl receptor.

VAIOTypewritten Text- KDEL sequence at the very end. It binds to a receptor that has the sequence KKXX, those proteins are going to get recognized by cop1 proteins.

Targeting of ER synthesized Proteins

Lysosomes

Acid hydrolases

Proton pumpH+-ATPase

Autophagy

Autophagolysosome

Residualbody

Targeting of Lysosomal Enzymes

Mannose

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VAIOTypewritten Text- lysoomes are ful of acid hydrolases that cut up proteins, sugars etc.- called acid hydrolases because they are active in an acidic environment, - what causes the acidity are other proteins called proton pumps, - major role of lysosomes in cells:

VAIOTypewritten Text- process of what lysosomes do. - Autophagy means to digest the old organelles that are no longer useful - ex. mitochondria that are old get digested inside the lysosome. ER forms around the old cell and form and autophago lysosome so it can digest the bbig organelles. When it's digested, a residual body is left. Some of the material gets expelled out of the cell. Sometimes we keep the materials and they're called lipofuscin. Old people have lot of lipofuscin in retina compared to young

VAIOTypewritten Text- how do we send the bad enzymes to the right place? Because of the carbohydrates.- some sugar monomers are called mannoses

Glycosylation of Lysosomal Enzymes

Cis Golgi

Mannose-6-phosphate


Mannose

Clathrin

Formation of Clathrin Coated Vesicles

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VAIOTypewritten Text- cisterna of the cis golgi- a lot of these enzymes are trans membrane.- N-acetyl... enzyme needs to see that there's a signal on the actual peptide, which says its meant to be sent to the lysosome. When it recognizes it, it will get some sugars (UDP, nucelotide sugars). Also a phosphate is added between sugar and monnose residue in position 6, that;s why its mannose-6-phosphate.-

VAIOTypewritten Text- in the cis golgi, the protein is tagged with the phosphae will get recognized by a receptor called mannose-6-phosphate receptor. that occurs at trans golgi- the protein coats that are made in the trans golgi are based on clathrin

VAIOTypewritten Text- clahtrin forms a coat- arf1 protein ,adaptor protein makes lin between the receptor- once clathrin leaves, coat is shedded, rab replaces


Mannose

Clathrin

Endosome

Targeting

Vesicle Fusion

Rabs

Tethering

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VAIOTypewritten Text- intermediate vesicle called endosome

VAIOTypewritten Text- how do

VAIOTypewritten Text- transport vesicle lost its coat and gets coated with rab (gtp bidning protein)- about 60 diff rabs known- between transport vesicel and target membrane there's another rab

VAIOTypewritten Text- first step is vesicle tethering (3 step process)

Rabs specify vesicle destination (targeting specificity)...

FYI!

Examples of Rabs (source: Wikipedia)

Docking

SNAREs

SNAREmotif

Fusion

NSF

SNAREs

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VAIOTypewritten Text- second step is called docking- it involves another protein SNAREs - three diff snares. - v-snares: vesicle snares- t-snares: traget snares 1) trans membrane snares 2) peripheral target snares- purpose of snares is to bring the vesicle as close as possible to the traget membrane. eventually the vesicle gets so close to target mebrane that they fuse together

VAIOTypewritten Text- ctyosolic protein NSF (adp depenedent protein) untwists snares so they can be used again

FYI!

Targeting/tethering: Rabs + tethering proteins Docking and fusion: SNAREs SNARE dissociation: NSF

Summary

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EndocyticPathway

Receptor-Mediated

Endocytosis

Coated pit

Coatedvesicle

Coated Pit Structure

Exterior ofcell

Interior of cell

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VAIOTypewritten Texttypes of endocytosis:1. receptor mediated : uptake of receptors and any material bound to them2. phagocytosis: uptake of particulate matter (cell eating)3. pinocytosis : uptake of solutes (cell drinking)- autophagy: the process of organelle turnover, is not a type of endocytosis per se but it uses a similar mechanism to phagocytosis

VAIOTypewritten Text- endocytic pathway:late endosome, mannose receptors get recycled,

VAIOTypewritten Text- clathrin is used as a coat to form the vesicles- coated pit: young vesicle formed at the membrane

VAIOTypewritten Text- clathrin can self assemble, will form the lattices

Clathrin

Triskelion

Animation of clathrin triskelionassembly into a lattice

Clathrin coated vesicle

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VAIOTypewritten Text- the shape of the clathrin molecule is called triskelion- clathrin molecule consists of 6 sub-units: 3 heavy chains, 3 light chains

VAIOTypewritten Text- form a soccer ball type structure- inside the cell, there's constraint. clathrin cell forms on surface of plasma membrane

VAIOTypewritten Text- ap2 is a multi subunit protein- has gtp binding protein called arf6

AP2 adaptorCellexterior

Cytosol

Dynamin

GTP hydrolysis

Conformational change

Endocytic PathwayHousekeeping

Receptors

TransferrinLow-density lipoprotein

Clathrin

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VAIOTypewritten Text- AP2 adaptor recognizes inter-cellular domain of the receptor. - there's a signal that says the receptor should be taken in- for signalling receptors, the signal is triggered by binding alot of material : down-regulation of receptors

VAIOTypewritten Text- once you have formation of the vesicle: - clathrin can' pinch off molecule on its own. Need dynamin (ring like protein) which forms a structure around the clathrin coated vesicle to pinch it off. Without the vesicle can't pinch off of the plasma membrane. Dynamin is a GTP binding protein. Binds: when it's bound to gtp, forms ring structure, need gtp hydrolysis for actual vesicle to pinch off and gets carried to where it's supposed to go. Eventually the clathrin coat falls off. experiment: putting a variant of GTPgammaS (doesn't hydrolized to GDP, stays in GTP forever). Noticed that you get rings (of gtp) forming because it can't pinch off. These are dynamin rings. Non-hydrolizable GTP analog > GTP gamma S. To ribosomes on ER when add GTPgammaS: they will always stay down to one another. To vesicles: the coat doesn't fall off, and the vesicle won't be able to bind to its target.

VAIOTypewritten Text1. housekeeping: receptors that are always brining in material that the cell needs to function normally. Can be used again. These receptors get recycled, and the decision to recycle or not takes place in the early endosome. They are vesicular in structure. Seperation of cargo (blue) and house keeping receptor because of pH. - examples: transferrin > brings iron in celllow-density lipoprotein > brings cholesterol

Endocytic PathwayHousekeeping

Receptors

TransferrinLow-density lipoprotein

Signaling Receptors

HormonesGrowth factors

Receptor down-regulation

Clathrin

Clathrin

Earlyendosomes

Lateendosomes

Endocytic Pathway

Posttranslational Protein Uptake

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VAIOTypewritten Text2. Signalling:hormones and growth signals really have to regulate the amount of signal that goes to the cell. Signalling receptors kill receptors when there's too much. - late endosome: if sent here, it gets sent to lysosome ... therefore whatever goes there is gone.

VAIOTypewritten Text- after translation- about how proteins get taken into the mitochondria-

Mitochondria

Targeting sequence Chaperone proteins Receptors Energy two sources

Mitochondrial protein import

Hsp60

Proton motive force

Brownian ratchet

Biaseddiffusion

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VAIOTypewritten Text- blue arrow: materials get taken in after translation

VAIOTypewritten Text1. targeting: different order of amino acids. The carrier proteins know that it is a protein going to the mitochondria2. chaperonses are necessary for bringing in protein once its docking on the receptor

VAIOTypewritten Text- signal sequence for proteins going into ribosome have no charge (hydrophobic)-here the signal sequnece is + charged and binds to receptor on what is called to TOM complex (trasport outermembrane complex). The cytosolic chaperones HSp70 bind to the protein that's being transported and use atp to mainatain the protein unfolded. Once protein binds to receptor : (left side of diagram) - the protein that gets completed incorporated into the matrix, the TOM complex sits very closely to th TIM 23 complex: pushing in the protein requires atp, to get the positibly charged tip of th protein past the intermembrane space because it is very positively charged (proton motor force). Once its past, th proton motor force actsas another energy force to get the protein in, will repulse positively charged signal (called pre-squence). Once the +charged signal gets passed, you get miochondrial chaperones. Once protein sticks head through TIM23, mito chap grab the head and brownian motion happens (molecules vibrate). Protein goes up and down and as soon as it goes down a matrix mito chap binds to it.

Mitochondria

Targeting sequence Chaperone proteins Receptors Energy two sources

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VAIOTypewritten Text- brownian ratchet moves in one direct. Once chaperone binds to it, it can't go back. Because it moves in one direction the protein diffues, but more in one direction than the other (biased diffusion). Once protein is all in mito processing peptidase finds sequnce. Right side: internal signal sequence that once it gets into TIM22, it will diffuse and will become part of the membrane




VAIOTypewritten Text2. chaperones are not just for folding.3. receptors on the TOM complex4. ATP and proton motive force for energy

biology 2020 section 3 (3pp)(1)

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