celll quiz 7

role of Golgi apparatus

membrane and protein trafficking in eukaryotic cells

cisternae

flatted membrane sacs that compose the Golgi

How many layers of cisternae make up the Golgi?

typically 3-8 but varies depending on cell type

Golgi stack

stack of cisternae

2 faces of the Golgi

• CIS face (entry), next to the ER

• TRANS face (exit), points towards plasma membrane

How do soluble proteins and lipids enter the cis Golgi network?

via transport vesicles dervied from the ER

proteins are transported through the Golgi from ___ to ___, and are sorted further at the ___

cis, trans, trans Golgi network (TGN)

common modification in the Golgi

• many of the sugar chains that are added in the ER (glycosylation) undergo further modifications in the Golgi

• more complex oligosaccharide side chains are added and removed by a series of enzymes that reside in sequence as the protein passes through the Golgi stack

order of travel through Golgi

cis Golgi network —> cis cisterna —> medial cisterna —> trans cisterna —> trans Golgi network

order of enzymes in the Golgi

mannosidase —> GIcNAc transferase —→ GaI transferase

anterograde transport

movement of material toward the plasma membrane

What events happen during anterograde transport?

• as a secretory granule fuses with the plasma membrane and discharges its contents (exocytosis), a bit of membrane from the ER becomes part of the plasma membrane

• vesicles can also bud to form endosomes that help make lysosomes

retrograde transport

flow of vesicles from Golgi cisternae back to the ER

purpose of retrograde transport

• allows the cell to balance the flow of lipids toward the plasma membrane

• also ensures a supply of materials for forming new vesicles

ER retention/retrieval sequences

short amino acid motifs found on ER resident proteins that ensure their return from the Golgi apparatus back to the Endoplasmic Reticulum (ER)

some proteins localized to the ER contain the sequence ___

RXR (Arg-X-Arg where X is any amino acid)

What is the classic retrieval sequence and what does it do?

• C-terminal KDEL sequence

• ensures that any ER resident proteins that might accidentally get packaged into vesicles and shipped to the Golgi will be captured and sent back to the ER

key elements in vesicular transport

• very regulated process

• selective - transport vesicles must carry only the particular content that they need to bring to the acceptor compartment (and not take what’s needed in the donor)

steps in vesicular transport

• sorting or selecting of cargo

• vesicle movement of vesicles along cytoskeletal elements (microtubules)

• tethering and docking onto the acceptor compartment membrane

• vesicle fusion with their acceptor compartment and release of contents

If the acceptor compartment is the plasma membrane, where will molecules be released in to?

the extracellular space

receptor mediated selection

specific soluble cargo molecules bind to transmembrane receptors on the luminal side, while the receptor’s cytoplasmic tail binds directly to coat proteins (like COPII or adaptors), concentrating the cargo into the vesicle

bulk flow

proteins can be trapped and packaged into vesicles randomly as the membrane buds, without direct binding to a specialized receptor

in vesicular transport, ___ carry material from one compartment to another

transport vesicles

donor compartment

compartment from which the material originates

target compartment

compartment to which the material is carried

How do transport vesicles find their way to the correct destination to deliver their content?

special proteins, including Rab and SNARE proteins, are responsible for interaction of transport vesicles with the correct target organelle

Rab proteins

• small GTPases that ensure vesicles fuse with the correct target membrane

• ensure that the transport vesicle fuses with the correct target membrane

• different Rab proteins, each specific for a particular type of vesicle

• each organelle and each type of vesicle carries a unique combination of Rab proteins, which serves as a molecular marker for each membrane type

tethering proteins

proteins that act as bridges between transport vesicles and target membranes to ensure correct docking and fusion

steps of fusion with target membranes

• tethering

• docking

• fusion

tethering (fusion)

• Rab protein on the vesicle is grabbed by a filamentous tethering protein that is part of the target membrane

• allows vesicle to dock on its particular target membrane

• SNARE proteins provide additional recognition

types of SNARE proteins and where they are located

• v-SNARE - vesicle SNARE, on the vesicle

• t-SNARE - target SNARE, on the target membrane

What are SNARE proteins?

transmembrane proteins that interact with one another to facilitate fusion of the vesicle with the target membrane

docking

• once the tethering protein has captured a vesicle by grabbing hold of its Rab protein, SNARES on the vesicle (vSNAREs) interact with complementary SNAREs on the target membrane (t-SNAREs), firmly docking the vesicle in place

• forms a trans-SNARE complex

fusion

same SNAREs involved in docking also play a central role in catalyzing the membrane fusion required for a transport vesicle to deliver its cargo

What does Botox have to do with SNAREs?

• Botox is produced by Clostridium botulinum

• SNARE proteins are essential for vesicle fusion

• in neurons, SNARE proteins allow synaptic vesicles to fuse with the plasma membrane and release ACh

• botox cuts SNARE proteins, which prevents vesicle fusion with the cell membrane and blocks ACh release

• muscle cannot contract —> temporary paralysis

vesicles that bud off from membranes have a ___ on their cytosolic surface and are therefore called ___

distinctive protein coat, coated vesicles

What happens to the protein coat on vesicles after budding?

the coat is shed, allowing the vesicle to interact with the membrane to which it will fuse

vesicle coats

cytosolic protein assemblies that drive membrane bending, cargo selection, and vesicle budding from donor membranes

coats are temporary: they assemble during ___ and are removed ___

budding, before fusion with the target membrane

3 major vesicle coats (what they are and what they do)

• COPII - mediates ER —> Golgi transport (anterograde)

• COPI - mediates Golgi —> ER and intra Golgi transport (retrograde)

• Clathrin - mediates endocytosis and Golgi —> endosome transport

ER —> Golgi transport uses ___ vesicles to….

COPII, deliver newly synthesized cargo

Golgi —> ER transport uses ___ vesicles with ___

COPI, retrieval signals

eukaryotic cells continually import materials by ___ and secrete intracellular materials by ___

endocytosis, exocytosis

How does endocytosis work?

material transported by endocytosis is captured from the external medium into a cell; cells can engulf vey large particles or even other cells by endocytosis

How does exocytosis work?

vesicles from inside the cell fuse with the plasma membane to release their contents into the external medium

structure of clathrin coated vesicles

an outer coat made up of the protein clathrin

steps of vesicle budding overview

• first there is a clathrin coated pit (invagination) in the plasma membrane

• clathrin molecules assemble into a basket like network

• starts to shape the membrane into a vesicle that then pinches off

What are clathrin molecules responsible for?

for shaping the membranes into vesicles, but not deciding which content will be transported inside the vesicle

adaptins

decide which content will be transported inside the vesicle

dynamin

assembles as a ring around the neck of each deeply invaginated coated pit, causing it to constrict

stages of vesicle budding in depth (roles of clathrin, adaptins, and dynamin)

• molecules for onward transport carry specific transport signals that are recognized by cargo receptors; adaptins help capture these cargo receptors by binding to them

• dynamin assembles as a ring around the neck of each deeply invaginated coated pit, causing the ring to constrict

• dynamin eventually causes the vesicle to pinch off from the plasma membrane

• after budding the coat is shed, allowing the vesicle to interact with the membrane to which it will fuse

3 different forms of endocytosis

• pinocytosis

• phagocytosis

• receptor mediated

pinocytosis

‘cellular drinking’, ingestion of fluid and molecules via small vesicles (<150 nm diameter)

phagocytosis

• ingestion of large particles (microoragnisms or cell debris) via large vesicles (>= 250 nm diameter), usually by specialized cells called phagocytic cells

• cells continuously ingest parts of their plasma membrane together with small volumes of extracellular fluid

receptor mediated

• cargo binds to specialized transmembane proteins called receptors

• allows internalization of material that can be concentrated many-fold, so that minor components of the extracellular fluid can be taken up without taking up correspondingly large volumes of fluid

pinocytosis is carried out mainly by ___ and ___

clathrin coated pits, vesicles

What balances pinocytosis fluid intake?

fluid loss during exocytosis

steps of receptor mediated endocytosis of cholesterol

• LDL particles bind to a particular transmembrane protein (‘LDL receptor’) molecules

• a clathrin-coated vesicle will form

• vesicle uncoats and moves towards an endosome

• the vesicle will fuse with an endosome and will release its content into it

• because the endosome has a low pH, the LDL receptor can no longer bind to LDL particles, so it releases them in the endosome

• the LDL particle is brought to lysosomes (major sites of degradation); LDL particles are degraded and free cholesterol is released

• cholesterol is membrane soluble so it gets out of the lysosome into the cytosol and can be used by the cells to incorporate into newly synthesized membranes

• the receptor is recycled - transported in a transport vesicle that will bud off from the endosome and will carry the receptor back to the plasma membrane, where it can be reused for another round of endocytosis

2 types of exocytosis

• unregulated/constitutive

• regulated/secretion

unregulated/constitutive exocytosis

• happens continuously without any special types of signals

• as soon as newly synthesized material arrives in the trans Golgi network, it is packaged into transport vesicles

• vesicles are carried to the plasma membrane to fuse with the plasma membrane and release their contents into the extracellular space

regulated exocytosis or secretion

• only happens when cells receive special types of signals and only in certain types of cells, specialized for secretion

• ex: cells that produce hormones, mucus or digestive enzymes

• material is stored in secretary vesicles and vesicles are stored until signal is received

insulin regulated exocytosis

• secretory vesicles store insulin in a pancreatic β cell

• insulin is released into the extracellular space in response to an increase in glucose in the blood