Unit 3 Bio 1020: Protein targeting, membrane trafficking, and signaling

Specific sugar added to asparagine (N) residue in ER lumen

Added to polypeptide, remain with the protein as it goes through membrane trafficking pathway, helps to prevent aggregation and eventually will be outside of cell. AKA N-linked glycosylation

Proteins are trafficked from the Golgi to

lysosomes or the plasma membrane

Protein __________ is maintained during membrane trafficking

orientation

___, _____, and ______________ are topologically like outside of cell

ER, Golgi, vesicle lumen

Vesicles bud from ______ membrane compartment and fuse to ______ membrane compartment

donor; acceptor

Organelle donor membrane budding off a vesicle containing cargo (i.e., integral membrane or luminal proteins) What are some donor organelles?

ER or golgi apparatus

Orientation is critical to _______

function

Vesicles budding requires ___________ that sculpt the membrane

coat proteins

What are the steps for coat proteins that sculpt the membrane?

1. Outer coat assembly and GTP hydrolysis acceleration

2. Coated bud formation

3. Vesicle scission, transport, and uncoating

Coat proteins then disassemble… (what happens after?)

releasing uncoated vesicle for trafficking to next destination

The endomembrane system is…

a group of membranes and organelles that function together as a coordinated unit (RER, Golgi, lysosomes, plasma membranes, all united by vesicle transport)

How do transport vesicles move?

Via motor proteins on microtubules and microfilaments of the cytoskelteon

Golgi Apparatus directionality

products from ER, in through the cis Golgi network, then moved out through trans Golgi network

What happens within the stacks of the golgi?

Enzymes within stacks motify proteins, especially by adding sugars

Sugar groups being added onto the chain by a series of enzymes located in

the Golgi lumen

What is the first step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

Incubate the professional secretory cells with pulse of radioactive amino acid

What is the second step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

Radioactive amino acid incorporates into newly synthesized polypeptides

What is the third step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

After fixing, embedding and thin sectioning, expose/coat thin section to film (silver halide crystals)

What is the fourth step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

Radioactive molecules emit beta-particles that ‘hits’ film, just like light

What is the fifth step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

Develop chemically, become dark silver grains

What is the sixth/last step of Jamieson and Palade’s experiment on the flow of traffic along the secretory pathway?

EM images with silver grains of cells at different time points

Exocytosis

seamless release of contents inside vesicle to the extracellular space

What do SNARE proteins do?

only function if bound in pairs (ex. v-SNARE and t-SNARE, one on transport vesicle and one on target membrane); execute fusion between vesicles and target proteins

How do membranes coalesce in fusion between vesicles and target membrane?

An influx of calcium after the transport vesicle docks allows for SNARE proteins to wrap around each other, pull apart the vesicle, and fuse the lipid bilayers

Endocytosis

delivering material into cells, budded off into a vesicle inside the cytosol

Endolysosomal system

• Recycle membrane receptors and lipids

• Degrade unwanted extracellular material or pathogens

• Extract nutrients from engulfed material

What is low density lipoprotein (LDL)?

a particle that carries cholesterol through the bloodstream to cells

There are pores in the nuclear membrane because

the double-layer lipid membrane acts as a barrier that prevents large, polar molecules and macromolecules—such as proteins and RNA—from passing freely in or out of the nucleus

Nuclear pores are semi-permeable to certain things because…

they are large enough to fit ions and small molecules and even small proteins (<40 kDa)

Selective transport of proteins into the nucleus import

• Transcription factors

• Histones

• Structural proteins

• RNA processing proteins

Selective transport of proteins into the nucleus exclude

• Nucleases

• Ribosomes

• Proteases

• Signaling factors (unless needed)

Selective transport of proteins into the nucleus export

• mRNA (spliced and checked)

• ribosomal RNA for assembly of ribosome in cytoplasm

• tRNA

Nuclear localization sequences (NLSs) allow proteins to be localized to the

nucleus

NLS is necessary for

transport of proteins into the nucleus

NLS is ______ for import of proteins into the nucleus

sufficient

GFP is ____ than the diffusion limit of nuclear pores

smaller

What mediates NLS?

Nuclear import receptors

What do nuclear import receptors do?

recognize and bind nuclear localization signals on proteins (‘cargo’)

Nuclear import receptors are similar to ____ as they recognize localization sequence

SRP (signal recognition particle)

How do nuclear import receptors interact and move proteins through the nuclear pore complex?

Nuclear pore complexes perforate the nuclear envelope

Disordered region of channel nucleoporins is made up of …

Phenylalanine (F) and glycine (G) amino acids repeats (FG repeats)

Nuclear import receptors do what to allow passage?

interrupt FG repeats on nucleoporins

How is cargo released from import receptor inside nucleus?

Another factor (Ran-GTP), concentrated in the nucleus, binds to the import receptor better than the cargo, then cargo is released

RanGTP provides _____ for transport

directionality

How is RanGTP maintained high in the nucleus?

When RanGTP goes out into the cytosol, RanGAP hydrolyzes RanGTP to Ran-GDP and can’t bind to import receptor, but then can diffuse back into the nucleus, turned back on by RanGEF (only in the nucleus), and maintains high concentration in nucleus

What is GTP?

Guanine triphosphate (similar to ATP) that can be hydrolyzed to GDP + Pi and regenerated

Proteins contain amino acids that serve as __________

zip codes

What are the membrane organelles part of the membrane trafficking system?

Golgi apparatus, endosome, lysosome, endoplasmic reticulum with membrane-bound ribosomes, plasma membrane

What are some final destinations of proteins (from the membrane trafficking system)?

Mitochondria, cytosol, peroxisome, nucleus

Typically, the “zip code” sequence is located near the (inside/outside) for cells to recognize where its destination would be

outside

Different __________ with specific features target to different destinations

sorting sequences

For protein signal sequences, many (but not all) targeting sequences are …

cleaved by peptidases after they reach their final destination

Proteins translated in cytosol will (go where?)

stay in cytosol or, if they contain a specific targeting sequence, will be targeted to destination (post-translational protein targeting)

What happens in co-translational protein targeting?

a protein is directed to its destination (such as the endoplasmic reticulum [ER] in eukaryotes or the plasma membrane in bacteria) while it is still being synthesized by the ribosome

The rough ER is studded with attached ______ that are actively translating proteins while ________ inserting them into the ER lumen. These proteins are then modified and shipped through the __________.

ribosomes; co-translationally; endoplasmic system

The golgi apparatus is the

protein sorting and shipment station

A common pool of ribosome subunits is used to …?

synthesize all the proteins encoded by the nuclear genome

A strand of mRNA is read …?

simultaneously by multiple ribosomes, each at a different stage of translation, with a growing polypeptide chain exiting from each one

What is a polysome?

multiple ribosomes all translating the same mRNA molecule at the same time. Each ribosome reads the same genetic message independently, moving from the 5’ end toward the 3’ end.

The ribosome closest to the 5’ start codon has the (shortest/longest) polypeptide?

shortest

The ribosome nearest the 3’ end has been translating the (shortest/longest) and therefore… ?

longest; carries the most complete chain

Which carries the most complete chain: the ribosome closest to the 5’ end or the 3’ end?

3’

What is the benefit of the polysome?

Greater protein output without the cost of making more transcript

Mitochondria have their own genome, but much of their genome was transferred to the ___________ over evolutionary time as they become more specialized. Therefore, many proteins for the mitochondria must be translated in the ________.

host genome; cytosol

Proteins must be ______ across or into membranes, which requires ___________ proteins or ____________

translocated; pore-like; protein complexes

_________ on destination membrane receive proteins and require a ___________ to import proteins through ____

Translocon pore; source of energy; pore

Different types of pores on nucleus, chloroplast, and peroxisomes, but all mediate…

translocation of proteins across or into membrane

If a protein’s targeting sequence were mutated, where would it end up, and why?

it would stay in the cytosol

There is a pore in the outer mitochondrial membrane that proteins pass through on their way in. If pore doesn’t close before or after a protein passed through, what would happen to mitochondrial function?

It could interfere with the protein gradient and therefore the electron transport chain, leaking the proteins out.

The lumen of the ER is like ?

extracellular space

What are the 3 different coat proteins?

1. COPII (ER to Golgi)

2. COPI (GOlgi to ER)

3. Clathrin (Golgi to plasma membrane to endosome)

What do coat proteins produce

force to deform donor membrane into bud and pinch

Coat proteins form a _____ around the vesicle membrane

cage

The ER is the ______________ step for the synthesis of proteins destined for secretion.

first and decisive

What shuttles cargo to and from one compartment to the next?

vesicles

What side of the golgi apparatus faces the ER?

cis

What side of the golgi apparatus faces the plasma membrane?

trans

What players are part of co-translational translocation (how ribosomes with polypeptide get targeted to ER surface)?

Ribosome-mRNA, polypeptide complex, ER signal sequence, translocon pore, signal recognition particle (SRP), SRP receptor

What is the signal recognition particle? What does it do?

A complex that recognizes a signal sequence on translating polypeptide and targets it to the ER membrane, allows for only the polypeptide to enter

How does the signal recognition particle (SRP) work at ER?

1. Translation begins in cytosol; ER signal sequences emerges from ribosome

2. SRP binds to ER signal sequence and ribosome

3. Bound SRP binds to SRP receptor which is integral to the ER membrane

4. SRP and Receptor release ribosome onto pore allowing insertion of N-terminal signal sequence into translocon pore, which is plugged until binding

5. ER membrane bound signal peptidase cleaves off N-terminal signal sequence

6. Co-translational insertion of the growing chain continues

7. Chain termination results in ribosome release from ER

8. Discharge of completed chain into ER lumen and closure of the translocon pore

Proteins are modified by (what molecules)?

sugars

Topologically, the lumen of the cell is like the _______ of the cell, facing ____ of the cell

exterior; outside

Why shouldn’t proteins flip orientations?

It would be energetically unfavorable, due to membrane and polar/nonpolar regions of protein

How does the sodium potassium pump work?

uses energy from ATP to actively move three sodium ions (Na+) out of the cell and two potassium ions (K+) into the cell

You know that the N-terminus of a lysosomal membrane proteins faces the cytoplasm, and that the GFP no longer fluoresces in the acidic lysosomal lumen. Where in the gene (5’ or 3’ end) would you fuse GFP to confirm it is on the cytoplasmic face—and what result would you expect to see in a fluorescent microscope if it’s on the correct side? What if it’s on the wrong side?

Place GFP on 5’ end because it is the N-terminus that faces the cytoplasm

You would see lots of fluorescence if it was on the correct side. You would not see fluorescence if it was on the incorrect side.

The lysosomal lumen is (acidic/basic)?

acidic

What does it mean for the GFP to be quenched?

No longer fluoresce

After coat proteins disassemble, releasing uncoated vesicle for trafficking to the next destination, what happens to them?

They are recycled for the next vesicle