Exam 3 - Cell Structure Part 1

Endoplasmic Reticulum

comprises a network of membrane that penetrates much of the cytoplasm (~50% of total membrane); highly dynamic; discovered in 1945 by Keith Porter, Albert Claude, Ernest Fullam

Smooth Endoplasmic Reticulum (SER)

short tubules
varies greatly in size in different cell types; will expand when needed

Rough Endoplasmic Reticulum (RER)

rough appearance due to the presence of ribosomes

flattened sacks

composition of the luminal or cisternal space is different from the surrounding cytosolic space
cytoplasmic entry point for proteins into vesicular trafficking system called biosynthetic pathway

protein synthesis on membrane bound-ribosomes vs free ribosomes

protein modifications

cell biology

study of the structures that make up a cell, how these structures function, form, and are maintained

Compartmentalization

Differences between prokaryotic and eukaryotic cells;
cellular compartment is (usually) a membrane encapsulated structure; means that proteins must be targeted to specific locations in the cell

Eukaryotic cells

cell that is highly compartmentalized;
A way to optimize cell behaviors and chemical reactions

• concentrate and isolate reactants
• optimize environment for reactions

routing possibilities for proteins

cytoplasm
nucleus
mitochondria
endoplasmic reticulum

sorting signals

amino acid sequences within proteins that direct protein localization and transport;
oligosaccharides attached to amino acids; Amino acids have different properties

proteins

_can passively diffuse to correct location in cell, or be transported within the cell

type of protein transport

gated transport
Translocator-based transport
Vesicular transport

gated transport

controlled opening of pore complexes; (Ex., selectivity of nuclear pore function in the nuclear envelope); folded proteins can go through, both passive and active transport

Translocator-based transport

transmembrane protein translocators transfer protein from one side of membrane to another. Protein must be unfolded to pass through.

Vesicular transport

often small spherical membrane packages that ferry proteins from one compartment to another

Requires:
-selection of cargo
-formation of a vesicle (budding)
-fusion of a vesicle with a target compartment

How do we identify what the signal sequence is for a given protein?

Molecular Biology approach
Biochemical approach
Genetic approach

Molecular Biology approach

Take bits of transported protein and fuse or "clone" amino acid sequence to a reporter protein and check distribution.A classic reporter gene… GFP

Biochemical approach

Direct purification of signal sequence

Genetic approach

Break (mutate) specific parts of protein to identify sequence for localization

Electron microscopy

First electron microscope invented in 1931
By the 1940s, cells were being stained with osmium and imaged with electron microscope

Why use electron microscopy?

Resolution of optical instruments is limited by the diffraction limit ~1/2 wavelength of light Electrons wavelength ~100,000x smaller than visible light

contiguous

ER and nuclear envelope are ___.

ER subcompartments

Smooth ER
Rough ER

Extracellular

outside the cell

cytoplasmic

Pertaining to cell matter (cytoplasm)

lumenal

Inside. Interior open space or cavity of a tubular organ (ER); becomes into cytoplasm once it leaves an organism

Smooth ER

functions in

• Ca2+ storage
• production of lipids, phospholipids and steroids (testestorne)
• detoxifying enzymes

proteins

_ need to be "targeted" to the right compartment

targeting sequence

___is part of protein itself

Membrane-bound ribosomes vs free ribosomes

no difference in the ribosomes per se
site of protein synthesis determined by amino-acid sequence at N-terminus of protein

Protein translocation into ER

signal sequence recognized by SRP
SRP binds to receptor
polypeptide moves into ER lumen through a protein-lined pore
movement can occur co-translationally or post-translationally

Steps of Protein translocation into ER

1) Binding of SRP to signal sequence; SRP RNA wraps around rRNA / proteins of ribosomes and pauses translation
2) Attachment of ribosome to ER membrane through the binding of SRP to the SRP receptor
3) Binding of SRP to protein translocator leads to release of SRP. Translation resumes, seal is tight between ribosome and translocator
4) Cleavage of sequence signal
5) Protein is released in ER lumen

Signal Recognition Particle (SRP)

Made of six proteins and one RNA (other example?)
Binds to signal sequences

1st Step of Protein Translocation into ER

Binding of SRP to signal sequence / translation stopped

2nd Step of Protein Translocation into ER

Attachment of ribosome to ER membrane through the binding of SRP to the SRP receptor

3rd Step of Protein Translocation into ER

Binding of SRP to protein translocator leads to release of SRP. Translation resumes, seal is tight between ribosome and translocator

4th Step of Protein Translocation into ER

Cleavage of sequence signal

5th Step of Protein Translocation into ER

Protein is released in ER lumen

Integral membrane proteins

proteins that are at least partially embedded in the plasma membrane;
Transmembrane proteins can have different topologies (orientations)

Translocation of lumenal/soluble protein

Signal sequence is recognized twice (by SRP and by translocator)
Signal sequence is 6-12 hydrophobic amino acids
Translocator is gated in TWO directions, cytoplasmic AND sideways bilayer opening

Start and Stop

____transfer sequences direct transmembrane proteins topologies

ribosome

SRP and its receptor interact to attach the __ to the ER membrane in close proximity to the translocon

lumen

Soluble proteins are released in the _ of the ER after cleavage of signal sequence by a peptidase.

start and stop

internal __ signal sequence are used for insertion of integral membrane proteins

integral membrane proteins

insertion of depends on the lateral gating of translocon

topologies

integral membrane proteins can have different ___

Protein modification in the ER

• Lipidation
• Hydroxylation of amino acids (collagen)
• Disulfide bond formation
• Glycosylation

Lipidation

addition of lipid to a protein
Example:

• GPI-anchoring

Hydroxylation of amino acids

addition of hydroxy group (-OH)
(e.g., collagen)

Disulfide bond formation

intramolecular or intermolecular covalent bond between sulfur groups in cysteine aminoacids

Oxidation reaction

Happens in the ER lumen but not in cytosol:

• oxidizing environment
• protein disulfide isomerase

Glycosylation

addition of carbohydrate (sugar) to a protein

N-linked glycosylation

Occurs in ER and Golgi

Starts in the ER:- a 14 sugar oligosaccharide is covalently attached

• transferred to Asparagine side chain (amide), therefore called ___

Continues in Golgi

N-linked glycosylation in the ER

Addition of glycans to a lipid carrier

• 2 N-acetylglucosamine
• 9 mannose
• 3 glucose
  Transfer to protein
  co-translational, within (N-X-S/T) sequence
  sugar transferred from lipid carrier

Oligosaccharyl transferase

enzyme that mediates transfer of assembled glycan
integral membrane protein with active site on lumenal side
one copy of enzyme per ER translocator

protein folding

unstructured chain of amino acid (inactive) to 3D folded structure (active)

what guides folding?

• all the information needed for the folding is contained in the amino acid sequence
• amino acids have different properties (charge, hydrophobicity)
• amino acids pack in such a way that the free energy of the protein arrives at a minimum

Protein quality control: protein folding

• glycosylation state
• chaperone proteins
• ER lumen environment

Protein quality control

Main components:
1) calnexin, a chaperone
2) glucosidase, enzyme that removes glucose
3) glucosyl transferase, conformation sensing enzyme, will transfer a glucose back if protein is misfolded
4) mannosidase, removes mannose.

Protein folding

___is reflected in glycosylation state

protein quality control

1) two glucose removed
2) remaining glucose binds to calnexin (chaperone), retains proteins in ER, give time to fold
3) glucosidase removes glucose, release protein
4) if protein is folded, it can leave ER. if not, conformation-sensing enzyme (glucosyl transferase) binds to misfolded protein and adds a glucose back.

cytoplasm

proteins that don't fold correctly are exported back into ___ and degraded

degraded

proteins that don't fold correctly are exported back into cytoplasm and ____

nonfolding protein

a slow-acting mannosidase enzyme that trims a mannose off the oligosaccharide tells the cell how long a protein has been in the ER
mannose-clipped protein can no longer be recycled and instead is sentenced to degradation
protein is exported from ER to cytoplasm
presence of N-glycosylation indicates to the cytoplasmic degradation machinery that the protein should be degraded.

mannosidase

glucosyl transferase is fastest enzyme, then export machinery, and finally _ is the slowest
the relative rates of the enzymes key for the system to work.

Unfolded protein response (UPR)

Under certain conditions, cells accumulates high levels of unfolded proteins in the ER

emergency action plan = _

• Decrease translation
• Increase ER
• Increase number of chaperones

If ___ is not sufficient, cell will trigger apoptosis

N-glycosylation

in the ER consists in the transfer of a 14 residues glycan

ER

Protein modifications occur at

lumenal

protein folding in the ER depends on environment, chaperones, glycosylation state

chaperones

protein folding in the ER depends on lumenal environment, _ , glycosylation state

glycosylation

protein folding in the ER depends on lumenal environment, chaperones, _ state

N-glycosylation

_ plays an important role in the protein quality control step (involving 4 main players)

Unfolded protein response (UPR)

Cells have an emergency action plan to deal with high level of unfolded protein, ___.

Membrane trafficking

Most trafficking steps require a vesicle

Vesicle

A membrane bound sac that contains materials involved in transport of the cell.

Cargo protein

protein transported by a vesicle

ER to Golgi transport

Combination of:

• retention system
• retrieval system

Retention system

mostly based on physical properties of proteins prevent them from entering transport vesicles (e.g., too large)
Some proteins escape => retrieval system as backup

retrieval system

The ER ____
ER proteins therefore have a sorting signal (or retrieval signal) that will signal they should be kept and/or returned to ER
ER resident soluble proteins have a retrieval signal (KDEL)

• KDEL Receptor (integral membrane protein)

KDEL

_ receptor must function differently between ER and Golgi

• must bind ___ proteins for retrieval in Golgi
• must not bind __- proteins in ER, and must release proteins when it returns them to ER

How does this occur?

• the ER has a neutral pH, while the Golgi has proton pumps that makes it a more acidic environment
• the__ receptor is very sensitive to pH
• is only active in acidic environment, so is active in Golgi
• is not active in neutral environment, so becomes inactive in ER, releasing proteins and not re-binding them

Golgi complex

Stacked structure, flattened, disklike cisternae
possesses a -cis and -trans face

• cis face (incoming vesicles from ER, outgoing vesicles to ER)
• trans face (outgoing to many destinations in cell, incoming from endosomes)

Golgi Complex function

functions

• protein post-translational modification
• protein sorting

Protein modification in golgi

Protein modifications

• phosphorylation (add phosphate)- glycosylation (N- and O-linked)
• sulfation (add sulfate)

Why glycosylate?

Not protein quality control in this case
1) Can target proteins to specific compartments (lysosome)
2) Glycosylation introduces special structural qualities to proteins

• sugars are some of the most rigid macromolecules
• keep protein from being attacked by protease
• keep bacteria from approaching cell surface

Trans Golgi Network

Sorting station for

• late endosome/lysosome
• early endosome/recycling endosome
• plasma membrane

ER to Golgi

Non ER resident proteins move from

retention and retrieval

o maintain unique compartmental composition, combination of system.

vesicular transport

During ___, cargo receptors bind to soluble cargo proteins

cis-Golgi

Proteins move from to trans-Golgi - two models

trans-Golgi

Proteins move from cis-Golgi to ___- two models

Proteins

get further modified in Golgi

Trans-Golgi network

acts a a sorting station

Steps in Vesicle Transport

1) Vesicle formation
a) cargo recruitment
b) budding
2) Vesicle scission
3) Transport and targeting
4) Tethering
5) Fusion

Vesicle formation

• coat and adaptor proteins
  Adaptor proteins bind cargo receptors / integral membrane proteins
  Adaptor proteins also recruit coat proteins
  Coat proteins help deform the membrane

3 major type of vesicles

• clathrin-coated vesicles
• COPI-coated vesicles
• COPII- coated vesicles

Clathrin

vesicles dedicated to trafficking from Golgi complex, plasma membrane, and some endosomal compartments

COPI-coated

vesicles dedicated to trafficking at the Golgi complex

COPII-coated

vesicles dedicated to trafficking from the ER

Clathrin

Stable basic unit is a triskelion
Triskelia then function as the basic unit for higher order ___ assembly (cage or lattice)
Coat property - reversible self-assembly

Vesicle scission

• Clathrin basket assembly cannot sever the lipid bilayer
• Dynamin is recruited to bud neck
• Dynamin cuts bud necks to free vesicle

Quickly after formation, vesicles loose their "coat"
This is the uncoating step

Transport and targeting

Rab proteins act as address labels for vesicles

• more than 60 known Rabs, different Rab proteins target different organelles
• Rab proteins bind:
• Motor proteins, mediate movement in cell
  -Tethering proteins, act to localize vesicle near target compartment

Rab proteins

___ act as address labels for vesicles
Different __ guide movement to different organelles

Vesicle tethering

Tethering proteins are bound to target compartment

• recognize incoming vesicle
• assemble into long, multi-protein complexes that reach into cytoplasm
• bind to Rab and localize vesicle near target membrane