BIOL10232 From Molecules to Cells

Cell Theory

1. Cells are the fundamental units of life

2. All organisms are composed of cells

3. All cells come from pre-existing cells

Unicellular organisms

A single cell carries out all the functions of life

Multicellular organisms

Made of many cells that are specialised for different functions (tissues and organs)

Central dogma

DNA makes RNA makes protein

Determining cell structure: Hooke

Used a primitive microscope to describe small chambers in sections of cork that he calls 'cells'

Determining cell structure: Leeuwenhoek

Discovered protozoa

9 years later, sees bacteria

Determining cell structure: Brown

Publication of observation of orchid, describes the nucleus

Determining cell structure: Schleiden and Schwann

Proposes the cell theory, stating that the nucleated cell is the universal building block of plant and animal tissues

Prokaryotes

No nucleus - DNA floats freely in the cell

No/limited internal membranes

Basic cytoskeleton

Eukaryotes

DNA in nucleus

Complex internal membrane system

Extensive cytoskeleton

Two domains of prokaryotes

Bacteria and archaea

Two domains of prokaryotes: bacteria

Streptococcus

E. coli

Salmonella

Treponema pallidum

Mycobacterium tuberculosis

Two domains of prokaryotes: archaea

Many live in hostile environments e.g. acidic hot springs, volcanic vents, marine sediment, cow's stomach

e.g. Thermus aquaticus

Examples of spherical bacterial cells

Streptococcus

Examples of rod shaped bacterial cells

E. coli

Salmonella

Examples of spiral bacterial cells

Treponema pallidum

Nucleolus

Production of ribosomal RNA and assembly of ribosomes

Organelles

Surrounded by one or more membranes made up of lipid bilayers - forms a physical barrier from the cytosol

Allows different protein contents and chemical environments to be maintained

Allows for each organelle to have a specialised function

Mitochondria

Site of oxidative phosphorylation

Generation of ATP

Double membraned organelle

Forms complex dynamic networks in the cell

Function of the extensively folded inner membrane of the mitochondtia

Increases the surface area for cellular respiration

Origin of membrane bound organelles (archaea to eukaryotes)

Bacterial ectosymbiont is enclosed by the fusion of the archaeal membrane forming a bacterial endosymbiont

The ectosymbiont moves to the cytosol

Formation of new intracellular compartments

Endoplasmic reticulum (ER)

Network of interconnected spaces enclosed by a single membrane that is continuous with the nuclear envelope

Makes secretory and membrane proteins and lipids

Entry point to the secretory pathway

Smooth endoplasmic reticulum

Abundant in human cells that is active in lipid metabolism

Abundant in liver for detoxification of lipid soluble compounds

Rough endoplasmic reticulum

ER derived calcium store in muscle cells

Important role in muscle contraction

Golgi apparatus

Receives proteins and lipids as cargo from ER

Cargo transits Golgi to plasma membrane

Modification of cargo (e.g. glycosylation)

Sorting of cargo to the correct location

Cytosol

Largest single compartment in the cell

Site of cellular processes e.g. protein synthesis and degradation and intermediary metabolism (e.g. glycolysis)

Location of the cytoskeleton

Model organisms: prokaryote

E. coli

Model organisms: lower eukaryote

S. cerevisiae (budding yeast)

S. pombe (fission yeast)

Model organisms: higher eukaryote

Vertebrate cells in culture

Model organisms: zebrafish

Vertebrate development

Model organisms: Drosophila melanogaster

Classical genetics

Development

Model organisms: Arabidopsis thaliana

Plant molecular biology and development

Model organisms: Caenorhabditis elegans

aka nematode worm

Genome sequencing

Programmed cell death

Key roles of ATP and GTP

Nucleotide binding controls protein shape, activity and function

Phosphorylation (phosphate group from ATP to serine/threonine/tyrosine to control protein function)

Phosphorylation in cellular processes

Cell growth

Gene expression

Cell survival

Cell cycle

Cell division

Metabolism

Function of lysosomes

Intracellular degradation

Function of endosomes

Sorting of endocytosed material

Function of peroxisomes

Oxidative breakdown of toxic molecules

The signal hypothesis

'Proteins have intrinsic signals that govern their transport and localisation in the cell' - Dr Gunther Blobel

These signals are specific sequences of amino acid in the protein

Signalling for targeting proteins to organelles

Signals act as intracellular postcodes

Target newly made proteins to the organelle/location where they function

Composed of specific sequences of amino acids

aka targeting signals/sorting signals/signal sequences

General rules for protein targeting

1. Signal to target the protein to the correct sub cellular destination

2. Receptor to recognise the signal

3. Transport machinery allowing proteins to cross the membrane

4. Energy (e.g. ATP/GTP

Signal sequence

Continuous stretches of polypeptide sequence made up of specific types of amino acids

Properties of amino acids

Determined by their side chains

Hydrophobic

Positive/negative charge

Specificity

Composition of the nuclear envelope

Two membranes: inner nuclear membrane and outer nuclear membrane

Underlying lamina

Nuclear lamina

Protein network made of lamins

Lamins

Type of intermediate filaments (forms the nuclear lamina)

Inner nuclear membrane

Contains proteins that bind to chromosomes and lamina

Outer nuclear membrane

Continuous with the ER (similar composition)

Nuclear pores

Over 30 different proteins

Gateway to the nucleus

Small water soluble molecules may pass through

Larger components (e.g. proteins/RNA) must be actively transported across the pores

Nuclear localisation signal (NLS)

Targets proteins to the nucleus

Short, less than 12 amino acids in length

Located anywhere in the protein

Not removed after the protein has been targeted

Common feature of the nuclear localisation signal (NLS)

High proportion of positively charged (basic) amino acids

i.e. Arg and Lys

Protein import into the nucleus

Receptor binds to proteins with NLS in cytosol

Fibrils direct the receptor to the pore

Receptors bind pore proteins

Cargo proteins moved into the nucleus

Size of nuclear pores

Large - folded proteins can be imported

Regulatory protein 1 (GAP) - aka Ran-GAP

Triggers GTP hydrolysis

Regulatory protein 2 (GEF) - aka Ran-GEF

Promotes exchange of GDP for GTP

Two conformations of GTPase Ran

GTP bound

GDP bound

Location of Ran-GDP

Cytosol

Location of Ran-GRP

Nucleus

Control of nuclear import by Ran

Prospective protein contains NLS and binds to the nuclear import receptor

Protein is moved through the pore via interactions with pore proteins

Receptor and cargo protein is imported into the nucleus

Ran-GTP binds the receptor and displaces the cargo protein from the receptor

Receptor bound to Ran-GTP is recycled back to the cytosol

Ran hydrolyses GTP and dissociates from the receptor

Mitochondria: matrix

Highly concentrated with enzymes, including those for the oxidation of pyruvate and fatty acids and for the citric acid cycle

Mitochondria: inner membrane

Folded into cristae

Contains proteins for oxidative phosphorylation

Contains transport proteins that move molecules into and out of the matrix

Impermeable

Mitochondria: outer membrane

Contains large channel-forming proteins (porins) so it is permeable to all molecules of 5000 daltons or less

Mitochondria: intermembrane space

Contains several enzymes which use ATP to phosphorylate other nucleotides

Contains proteins which are released during apoptosis

Mitochondrial targeting signal sequences

High content of Arg & Ser/Thr

N-terminus

20-80 amino acids long

Cleaved after import into the mitochondrial matrix

Tends to form an alpha helix with positively charged residues and negatively charged residues. As a result, the sequence of the signal and its secondary structure are both important

Chaperone proteins

Hsp70s

Hydrolyses ATP to drive mitochondrial protein import

Pulls the protein into the matrix and helps refold it

Distribution of ER in different cell types

Amount of smooth and rough ER varies in different cell types

Depends on the requirement for protein and lipid synthesis

e.g. pancreatic cells have extensive rough ER; hepatocytes have extensive smooth ER

Proteins entering the ER: soluble proteins

Water soluble

Secreted from cell into extracellular environment by secretory proteins

Located in the lumen of organelles of the endomembrane system

Examples include insulin, digestive enzymes, lysosomal enzymes

Examples of organelles in the endomembrane system

Endoplasmic reticulum

Golgi apparatus

Endosomes

Lysosomes

Proteins entering the ER: integral membrane proteins

Hydrophobic sequences which form membrane spanning domains

Embedded into the lipid bilayer

Membranes of the endomembrane system, plasma membrane (NOT mitochondria)

Examples include membrane receptors, channels, transporters

Endoplasmic reticulum targeting signals

aka ER signal sequence

Stretch of 8+ hydrophobic amino acid residues near the N terminus

Targeting to the ER: role of signal recognition particle (SRP)

Located in the cytosol, SRP binds to the ER signal sequence, pausing translation

SRP binds to the SRP receptor on the ER membrane and Sec61 (protein translocator)

SRP is displaced and released for reuse

The ribosome passes to the translocator; translation resumed

Release of soluble proteins into the ER lumen

The ER signal sequence binds to the Sec61 translocator causing the channel to open

The polypeptide is threaded through the channel as a loop while translation is occurring (co-translational translocation)

Protein translocator is closed

Signal peptide is cleaved by signal peptidase

Mature protein is released into the ER lumen

Translocation

Movement of proteins across the ER membrane

Co-translational translocation

Translocation occurs at the same time as translation

The protein translocator: Sec61 channel

Multi-subunit complex of proteins forming a channel in the ER membrane

Allows the protein chain to be translocated across the ER membrane through a central pore

Pore is closed in the absence of translating polypeptide

Protein translated in vitro

Cell extract (contains ribosomes, cytosolic factors)

mRNA

Radioactive amino acids

Organelle membranes (ER, mito)

Cell free assay

Easy to manipulate

Different mRNA

Add/remove soluble components

Protein folding in the ER

After translocation, the partially/loosely folded polypeptide chain must fold into the correct 3D conformation in the ER lumen

Molecular chaperones assists folding

BiP (an ATPase) binds exposed hydrophobic residues

Calnexin binds N-glycosylated proteins

Anchoring of transmembrane proteins in the lipid bilayer by hydrophobic sequences

Signal sequence binds Sec61 causing the channel to open

The hydrophobic stop-transfer sequence enters the translocator and stops the movement of the polypeptide through the channel

The stop-transfer sequence is released from the channel into the bilayer forming a transmembrane domain - signal sequence is cleaved

RESULT: the protein is inserted into the bilayer; fixed orientation; N-terminus in the lumen

Protein modification in the ER

1. Cleavage of signal sequence

2. Formation of disulphide bond (oxidation)

3. Glycosylation - covalent attachment of carbohydrate

Protein modification in the ER: disulphide bond formation

Formed by oxidation of cysteine side chains

Stabilise folded structure of proteins

Catalysed by protein disulphide isomerase inside the ER lumen

ER lumen is oxidising

N-linked glycosylation

N = Asparagine

Oligosaccharide transferred to the protein from a dolichol (lipid donor)

Catalysed by oligosaccharyl transferase (OST)

Functions of N-linked glycosylation

Assists protein folding

Modified to create mannose-6-phosphate tags which act as a lysosome sorting signal

Acts as a ligand for specific cell-cell recognition events

Role of N-linked glycosylation in the glycocalyx

Eukaryotic cells are coated in carbohydrates attached to proteins and lipids called the glycocalyx - forms a protective layer

Made at the ER and Golgi before delivery to the plasma membrane

Secretory pathway

Vesicular transport pathway that moves proteins and lipids from the ER through the Golgi to the plasma membrane/extracellular space

Allows material to move outwards (ER to Golgi to plasma membrane/endosomes/lysosomes)

Transport vesicles

Transports proteins and lipids that are destined to the Golgi, lysosomes and plasma membrane

Membrane lipid synthesis

Enzymes on the cytosolic face of the ER membrane synthesise new phospholipids

Scramblase transporters transfer phospholipids between leaflets of the membrane non-selectively

Symmetric growth of both halves of the bilayer

Endocytic pathway

Vesicular transport pathway that moves protein and other molecules from the plasma membrane/extracellular space into the cell interior

Allows material to enter the cell

Mediation of vesicular transport by coat proteins: endoplasmic reticulum

COPII

Mediation of vesicular transport by coat proteins: Golgi apparatus (Golgi cisternae)

COPI

Mediation of vesicular transport by coat proteins: early endosome/trans Golgi network

Clathrin

Coat proteins: clathrin

Functions at the plasma membrane and Golgi

Forms a highly ordered basket-like structure

Shapes the membrane

Captures cargo e.g. molecules to be taken into cells from the plasma membrane or outside the cell

Cargo selection: adaptins

Helps clathrin attach to the membrane forming clathrin-coated pit on the cytosolic face of the membrane

Binds cargo receptors which recognise specific sorting signals on cargo proteins, recruiting them into the vesicle

Vesicle budding

Clathrin assembles into cage, causing the membrane to invaginate into a bud

Dynamin assembles as a ring around the neck of the bud

Dynamin

Is a GTPase

Conformation of dynamic changes when it hydrolyses GTP

Together with other proteins, this constricts the neck of the bud

Budding vesicle pinches off

Mutant dynamin causes paralysis in flies

Temperature sensitive mutant in Drosophila

Vesicles cannot pinch off from the plasma membrane

Results in the loss of neurotransmitter vesicles in synapses

Vesicle uncoating

After budding, coat proteins are removed (i.e. uncoating)

Requires molecular chaperones and ATP

Produces naked transport vesicle that can fuse with the target membrane

Vesicle docking and fusion: Rab proteins and tethers

Rab protein on the vesicle binds to tethering protein on the target membrane

Specific pairs of Rabs and tethers - docking

Initial recognition between vesicle and target membrane - fusion

Vesicle docking and fusion: SNAREs

Catalyses the fusion of vesicle and target membranes

SNARE proteins in vesicle and target membrane

Complementary pairs v-SNAREs and t-SNAREs interact, docks vesicle onto the target membrane

v-SNAREs on vesicle

t-SNAREs on target membrane

SNAREs catalyse the fusion of vesicle and target membrane

Bilayers must be brought within 1.5 nm for lipids to fuse

t-SNAREs and v-SNAREs wrap tightly around each other which pulls the membrane together to drive the final fusion event

Water must be displaced => energetically favourable

May require an additional signal

1. Transport vesicle docks

2. Membranes coalesce

3 Lipid bilayers fuse