Cell Bio chp. 15 - Intracellular compartments and Protein Transport

What occurs in different membrane enclosed compartments within the cell

Different metabolic processes

Organelles surrounded by at least one membrane and function

• nucleus

• Rough ER

• Golgi

• Mitochondria

• lysosome

• endosome

• peroxisome

Where you may find some cell components (In class)

• Enzymes for Krebs - Mitochondria

• Enzymes for photosynthesis - Chloroplast

• Enzymes for unraveling DNA, DNA replication - Nucleus

• Enzymes that are receptors - cell membrane

• Enzymes for detoxification - lysosomes and peroxisomes

• Cytoskeleton - Cytoplasm

How do proteins move (In class)

• proteins traffic naked

• pack proteins into vesicles

Where are all proteins made (In class)

They are all translated in free cytoplasmic ribosomes

(Later flashcards mention either continuing translation in the cytoplasm or going to the ER)

Path of proteins traveling in ribosomes (In class)

Translation starts in cytoplasm then moves to ER where it can be transported as a vesicle

Path of naked protein travel (In class)

starts in cytoplasm and gets to translocators except for peroxisomes and nucleus

Liver cell name

hepatocyte

Volume and number per cell of membrane enclosed organelles within liver cells

Do not need to remember specific numbers. Just know generally which make up majority of cell

Two ways membrane bound organelles evolved

• invagination: Cell membrane folded in on itself to form a membrane within the cell (Formation of nucleus, ER, and Golgi from ER folding itself)

• Endosymbiosis theory: pre-eukaryotic cells engulfed other prokaryotic cells to create membrane bound organelles (formation of mitochondria and chloroplast)

3 mechanisms in which proteins are transported into organelles

• 1) Transport through nuclear pores (entering nucleus)

• 2) Transport across membranes via protein translocators guiding unfolded proteins across membranes(entering mitochondria/chloroplast)

• 3) Transport by vesicles (ER → Glogi)

(Peroxisomes can do vesicles and translocators)

Signal sequence

Amino acid sequence that directs protein to a specific location/compartment within the cell.

Which side of polypeptide is signal sequence towards

N-terminus

How many amino acids typically make up a signal sequence

15-16 amino acids

Features of signal sequences that determine which organelle they will go to

• length of sequence

• amino acid properties

what to remember for sequence that directs to nucleus

Has a bunch of lysine’s (Lys)

signal sequence for protein going to cytoplasm

Does not have a signal sequence

Signal sequence experiment

ER protein with an ER signal sequence resides in the ER while a cytosolic protein without a signal protein resides in the cytosol. Genetic engineers can remove the signal sequence and attach it to the cytosolic protein so that the ER protein is in the cytosol and the cytosol protein is in the ER.

Nuclear envelope

double membrane that encloses nuclear DNA and is made up of the inner nuclear membrane and outer nuclear membrane

Inner nuclear membrane

contains proteins that are binding sites for chromosomes and proteins that anchor nuclear lamina

nuclear lamina

mesh of protein filaments lining the inside of the inner membrane to provide structural support.

(the cell cortex of the nucleus)

outer nuclear membrane

resembles the ER and is continuous with the ER membrane

nuclear pores

penetrate the nuclear envelope and can allow proteins to enter the nucleus. Is specific to what is allowed in.

nuclear pore structure

• cytosolic fibrils: fibrils on outside of nuclear envelope

• nuclear basket: fibrils on nuclear side form a basket structure

• pore complex proteins: Proteins within the membrane that create a gel like structure with fibrils that allow for specified molecules to enter

Nuclear transport steps

• cargo proteins contain a nuclear localization signal that is recognized by a nuclear import receptor

• nuclear import receptors guide the protein to a pore by interacting with cytosolic fibrils

• nuclear import receptors along with the protein go through the unstructured, specific pore protein region until entry where the protein is released

• nuclear import receptors go back into the cytosol through pores to be reused

Do proteins change structure when passing through nuclear pores into the nucleus

Proteins structures remain unchanged

(Only organelle where proteins do not need to unravel to pass through)

Is energy needed for proteins to go through nuclear pores

Yes, GTP is used

Nuclear import receptor process with GTP

• After entering the nucleus with a protein, Ran-GTP binds to the receptor and facilitates the release of the protein.

• The receptor is transported back into cytosol through a pore, still bound to Ran-GTP

• In the cytosol, Ran hydrolyzes GTP and Ran-GDP and Pi detach, allowing the nuclear import receptor to bind to another protein

What must proteins do before entering the mitochondria and chloroplast

They need to unfold

Mitochondrial precursor protein entry process

• Precursor proteins signal sequence binds to a receptor protein on the outer membrane

• The receptor is associated with a translocator protein (TOM) that transports the signal sequence from the outer membrane to the intermembrane space

• The complex of signal sequence, receptor, and TOM diffuses laterally until the signal sequence is recognized by a second translocator protein (TIM)

• The two translocator proteins diffuse the protein across the outer and inner membrane while unfolding the protein

• When inside the matrix a signal peptidase will cleave off the signal sequence

• Chaperone proteins will refold proteins in the matrix using the hydrolysis of ATP

Why must proteins unfold when passing through translocators

Translocators have a narrow opening

Two places proteins can enter peroxisomes from

Cytosol and ER

(Only organelle that can allow proteins to enter through translocators and vesicles)

Process of Protein transport from cytosol to Peroxisome

• Peroxisome proteins contain three amino acid signal sequences that are recognized by receptor proteins in the cytosol

• The protein is delivered and enters the peroxisome by the receptor protein that then returns to the cytosol after

• There are also translocator proteins on the peroxisome protein but these do not unfold the protein

Process of protein transport from ER to Peroxisomes

Vesicular transport

• Bud off ER and fuse with Peroxisome membrane

• Or become Peroxisomes themselves

Zellweger syndrome

abnormalities in the brain, liver, and kidney due to not being able to detoxify

will not make it past 6 months

Zellweger syndrome causation

Mutations that block peroxisome protein import due to lack of translocators

What happens to polypeptides (proteins) as they are transferred to the ER

They are simultaneously being synthesized

Where do proteins with no ER signal end up

In the cytosol

How do ribosomes end up on the ER to make up the rough ER

they are dragged onto the ER surface by the signal sequence of a polypeptide it is translating

Polyribosome

Multiple ribosomes binding and translating a single mRNA

Process of ER protein entering ER

• Ribosome in a pool of subunits within the cytosol begins translating mRNA

• If the protein has an ER signal sequence it will guide the ribosome to the ER

• The proteins signal sequence will associate to a translocator on the ER membrane where it can enter the ER lumen

multiple ribosomes may begin synthesizing the same mRNA sequence (polyribosome)

Do proteins need an energy source to enter the ER

No, they are translocated as they are being made so no additional energy is needed

SRP

Signal recognition particle

Soluble protein transport into the ER process (More detailed process)

• SRP binds to both the ER signal sequence and the ribosome, slowing protein synthesis

• The SRP-ribosome complex (SRP, ribosome, and polypeptide) then binds to an SRP receptor in the ER membrane

• SRP is displaced and can be reused

• ribosome and ER signal sequence are passed to a protein translocator in the ER membrane

• Growing then proceeds normally and the translocator transfers the newly synthesized protein into the ER lumen as a loop

• A signal peptidase will eventually cleave the signal sequence from the polypeptide

• The signal sequence stays in the bilayer and is degraded

• The finished soluble protein enters the ER lumen and the translocator closes

What determines the arrangement of proteins in the lipid bilayer

Start and stop signals

hydrophobic Stop-transfer sequence

hydrophobic start-transfer sequence

(Not soluble)

Transport to the ER of transmembrane proteins

• Start-transfer sequence binds to a protein translocator initiates transfer

• when a stop transfer sequence enters the translocator protein, both sequences are transferred to the lipid bilayer

• a signal peptidase will cleave off the start-transfer sequence

• A transmembrane protein anchored by the stop-transfer sequence is left

Side that N-terminus and C-terminus face for transmembrane proteins

• N-terminus: Faces lumen and outside of cell

• C-terminus: Faces cytoplasm always

Inward endocytic pathway

• Endocytosis ingests extracellular molecules in vesicles derived from plasma membrane

• Vesicles are delivered to early endosomes then late endosomes and finally lysosomes

Outward secretory pathway outcome 1

Proteins from the ER pass through the Golgi to early endosomes then to late endosomes and finally a lysosome

Outward secretory pathway 2

Proteins from ER go through Golgi and are then passed to the plasma membrane where they go through exocytosis

Can proteins enter the Golgi from the cytosol directly

No, must come from ER or plasma membrane as a vesicle

Protein coated vesicle

membrane enclosed vesicles are surrounded by proteins on its cytosolic surface formed by budding off protein coated regions

clathrin

Protein that makes up coat of transport vesicles budding off the Golgi (outward secretory pathway) or the plasma membrane (inward secretory pathway)

Dynamin

GTP binding protein that closes the coated vesicle when budding off membranes

Adaptins

assemble clathrin on surface of vesicles and help capture cargo molecules and bind them to cargo receptors

transport signals on cargo molecules

Protein coated vesicle formation process

• Adaptin binds to a cargo receptor with a cargo molecule which binds adaptin to cytosolic exterior of membrane

• vesicle formation begins and dynamin wraps around budding vesicle neck

• Dynamin will hydrolyze its GTP and other proteins will help to pinch of vesicle

• when the vesicle is formed the protein coat of clathrin and adaptin leaves

• the naked transport vesicle can fuse with its target membrane

Types of coated vesicles

• Clathrin coated (From Glogi): made up of clathrin and adaptin 1 and end up at the lysosome via endosomes

• Clathrin coated (From Plasma Membrane): made up of clathrin and adaptin 2 and end up at endosomes

• COP-coated: Come from ER, Golgi cisterna, or Golgi apparatus, made of COP proteins, and arrive at the Golgi apparatus, Golgi cisterna, or ER (respectively)

What does vesicle docking depend on

Tethers and SNAREs

Rab proteins

GTP-binding proteins present on surface of transport vesicles and organelles to serve as a marker that ensures transport vesicles fuse with the correct membrane

tethering proteins

Filamentous transmembrane protein involved with docking of transport vesicle with target membranes