How do proteins know where to go in the cell?

Signal hypothesis

A theory proposing that proteins have a signal sequence that directs them to their correct cellular compartment, such as the nucleus, ER, or mitochondria.

Signal sequence

A short peptide sequence that directs the protein to a specific cellular location, such as the nucleus, mitochondria, or ER.

Necessary

A condition that must be present for a certain event or process to occur. In the context of signal sequences, removing the signal sequence and seeing if the protein still localizes properly is testing if it’s necessary

Sufficient

A condition that is capable of producing a certain effect on its own. In the context of signal sequences, adding that signal sequence onto another protein and seeing if it’s enough to make that protein localize to where the signal sequence tells it to is showing sufficiency

post-translationally

Nuclear proteins are transported into the nucleus _________

Nuclear envelope

The double membrane surrounding the nucleus that separates the contents of the nucleus from the cytoplasm.

Nuclear pore

Large protein complexes embedded in the nuclear envelope that allow the regulated exchange of molecules, such as RNA and proteins, between the nucleus and the cytoplasm.

Contiguous

The nuclear membrane is _____ with the ER

ions, small molecules, ATP, GTP

Molecules that can move freely through the pores via passive diffusion

Proteins, larger molecules, RNAs

Molecules that need to move in energy dependent mechanism across a nuclear pore

40kD

proteins larger than _____ need to move in energy dependent mediated transport across nuclear pores

FG-repeat domains of FG nucleoporins

Hydrophobic regions found in nucleoporins on the cytosolic side of the nuclear pore that create a hydrophobic meshwork. which restricts the passive diffusion of larger molecules, such as proteins and RNA, across the nuclear envelope

Nucleoporins

Proteins that make up the nuclear pore complex, NPC

Nuclear Localization Signal (NLS)

A peptide sequence in a protein that acts as a signal for import into the nucleus.

Nuclear Export Signal (NES)

A signal sequence that directs proteins or RNA from the nucleus to the cytoplasm, tends to be hydrophobic AA

Positive

What charge is the string of AA that serves as a NLS?

G-protein

A protein that acts as a molecular switch inside cells, toggling between an active (GTP-bound) and inactive (GDP-bound) state.

Ran

A small GTPase involved in nuclear transport, particularly in the exchange of proteins and RNA between the nucleus and the cytoplasm.

RAN-GAP (Ran GTPase Activating Protein)

A protein that activates the GTPase activity of Ran, causing it to hydrolyze GTP to GDP, thus switching Ran to its inactive form.

Ran GEF (Ran Guanine Exchange Factor)

A protein that facilitates the exchange of GDP for GTP on Ran, activating Ran and allowing it to bind to cargo for nuclear transport.

Importin

A protein that binds to cargo proteins containing a NLS and facilitates their import into the nucleus through the nuclear pore.

Importin

_______ can interact with the FG repeat domains on the nucleoporins

Cargo complex

Firstly, importin binds to the cargo, creating the ______

Nucleoplasm

After importin binds to the cargo in the cytoplasm, they go through the pore to go into the ______

Ran GDP

As the cargo complex goes through the pore from the cytoplasm, _____ is also moving the same direction

Cytoplasm

Where RAN-GAP localizes

Nucleus

Where RAN-GEF localizes

RAN GDP

Becuase RAN-GAP localizes in the cytoplasm, there is a lot of _____ too in the cytoplasm

RAN GTP

Becuase RAN-GEF localizes in the nucleoplasm, there is a lot of _____ too in the nucleoplasm

RAN GEF, RAN GTP

Now that both the cargo complex and RAN GDP have made it into the nucleoplasm via the pore, _____ turns the RAN GDP into ______

importin, RAN GTP

_____ has high affinity for _____ which is important for when they’re both in the nucleoplasm

cytoplasm

Once RAN-GTP and Importin bind in the nucleoplasm, they head back to the _______ after sucessfully dropping off the cargo

RAN GAP

Once the RAN-GTP/Importin complex arrives back in the cytoplasm, ________ causes a conformational change and unbinds the two

Exportin

A protein that binds to cargo proteins with an NES and facilitates their export from the nucleus.

RAN GTP

Exportin will only bind to cargo when associated with ________

Ran GTP, exportin, cargo

When utilizing a NES, the cargo complex that moves to the cytoplasm consists of ________

RAN GAP

Once the cargo complex is exported out of the nucleoplasm into the cytoplasm, it comes apart when _____ causes a conformation change in RAN GTP

Secretory pathway

Virtually all of the entire cell/plasma membrane are synthesized via this pathway

rough ER

pancreatic cells have a lot of _______

Pulse chase experiments

• A method used to study the dynamics of protein synthesis and trafficking, where cells are first exposed to a labeled precursor (pulse) and then followed over time to see where the protein moves (chase).

secretory pathway

The pathway through which proteins are synthesized in the ER, processed in the Golgi, and secreted out of the cell or delivered to other organelles.

radiation sensitive emulsion (silver)

To visualize the results of the pulse chase experiment, you fix the cells, and then dip the grid in _________ to visualize with TEM

Cell-Free System

A laboratory method used to study protein synthesis or other biochemical processes using a cell extract that contains the necessary machinery (ribosomes, enzymes, etc.) but no living cells.

vesiculates

When you homogenize cells, the ER ________ instead of retaining its shape as long cisternae, allowing you to isolate microsomes containing protein synthesis machinery

Equilibrium density gradient centrifugation

When preparing cell free systems, this technique allows you to separate your “smooth” microsomes from your “rough” ones

protease treatment, mild detergent, SDS-PAGE

How to tell if protein has entered an ER microsome?

co-translationally

The import of protein into ER happens ______ in contrast to nuclear transport

Co-Translational Import into ER

The process by which proteins are synthesized on ribosomes and simultaneously imported into the ER during translation.

Signal Recognition Particle (SRP)

A protein-RNA complex that recognizes the signal sequence of a nascent protein and directs the ribosome to the ER membrane for co-translational import.

SRP Receptor

A receptor in the ER membrane that interacts with the SRP-ribosome complex, allowing the nascent protein to be inserted into the ER.

stops translation

SRP _____ ______ when it binds to the signal sequence

ER translocon

The interaction of SRP with the SRP receptor allows the ribosome to bind to the ________

G-proteins

SRP and SRP receptor are both _______

GTP hydrolysis, translocon, translation

When ________ occurs, SRP is released and the ribosome “sits” down on the _______, and _____ resumes

Stop Transfer Anchor Sequence (STA)

A hydrophobic sequence in a nascent protein that causes the protein translocon to stop translocating the protein into the ER, anchoring it in the membrane.

Signal peptidase

This protein in the ER lumen cleaves the signal sequence from the nascent polypeptide

cytosol

Positive strings of AA in front of or behind internal signal-anchor sequences always are in the ________

Signal Anchor Sequence (SA)

A hydrophobic sequence in a protein that directs it to be inserted into the membrane without stopping translocation, allowing the protein to span the membrane multiple times.

SA-II

Internal signal-anchor sequence that has positive AA on side facing N-terminus

SA-III

Internal signal-anchor sequence that has positive AA on side facing C-terminus

ER Lumen

The inside space of the endoplasmic reticulum, where proteins undergo folding and post-translational modifications like glycosylation.

Rough ER post-translational modifications

1. Formation of disulfide bonds

2. Proper folding

3. Addition and processing of carbohydrates

4. Specific proteolytic cleavages (cleaving N-term signal sequence)

   1. Assembly into multimeric proteins

Protein Disulfide Isomerase (PDI)

An enzyme in the ER lumen that catalyzes the formation and isomerization of disulfide bonds, helping proteins achieve their correct 3D structure.

ER Lumen

Where is Protein Disulfide Isomerase (PDI) found?

reduced, oxidized

After the disulfide bonds are formed, the PDI gets ____ and the substate protein becomes _____

N-linked Glycosylation

The process by which a sugar is added to the “N” of an asparagine R group in a protein, typically in the ER.

O-linked Glycosylation

The addition of sugar to the “O” of serine or threonine residues in proteins, usually occurring in the Golgi.

Glycosyl transferases

Enzymes that catalyze the transfer of the 14 sugar group to asparagine, can occur before/after synthesis is completed

Glucose residues

In the sugar complex added to asparagine by glycosyl transferase, ________ are important for proper protein folding in the ER

dolichol phosphate

Process of N-linked sugars begins with lipid carrier __________ embeded in ER membrane. Next, precursor oligosaccharide (14 sugar) is built on the _______ in stepwise manner. After, glycosyl transferase moves the sugar to the Asn

Calnexin

A chaperone protein in the ER that binds to partially folded proteins to aid in their proper folding and prevent aggregation.

2 of the 3 glucose subunits

Removal of _________ allows calnexin to bind/facilitate folding because the singular gluocse enables calnexin to bind to the nascent polypeptide

Unfolded protein response

A stress response triggered by the accumulation of misfolded proteins in the ER, leading to increased protein folding capacity and degradation of misfolded proteins.

Cis golgi network

large tubulovesicular network that connects to the cis face of the Golgi stack and receives and processes biosynthetic output from the ER

Cis cisternae

flattened, disc-shaped sacs that are closest to the endoplasmic reticulum (ER)

Medial cisternae

The middle region of the golgi, between the cis and trans

Trans cisternae

the outermost, furthest cisternae in a Golgi stack that face the plasma membrane

Trans Golgi Network (TGN)

The region of the Golgi where vesicles are sorted and directed to their final destination, such as the lysosome, plasma membrane, or secretory vesicles.

Vesicular Transport Model

A model that suggests that vesicles carry cargo between different Golgi cisternae.

Cisternal maturation model

A model that proposes that the Golgi cisternae themselves mature, moving from the cis to the trans face while carrying cargo along. Resident enzymes travel “backwards” through vesicles

yeast mutants

Analysis of ________ has defined the major steps/players in the secretory pathway

Advantages of yeast

1. Haploid, only one copy of gene. easier to mutate one copy than worry about if second copy is covering up any potential phenotype

2. Easy to perform large scale screens to isolate termperature sensitive (ts) mutations

3. Conservation of process in eukaryotes

permissive temperature

Temperature that mutant is usually grown at, where the protein that’s been mutated is still functional

Restrictive temperature

Temperature where protein bearing mutation now becomes dysfunctional

COPII, COPI, Clathrin

What is the mechanism by which vesicles form/cargoes are selected?

Coat proteins

Proteins that form a coat around vesicles, which helps in their budding and cargo selection for membrane transport. Examples include COPI, COPII, and clathrin.

COPII

A protein complex involved in anterograde transport from the ER to the Golgi apparatus

COPI

A protein complex involved in retrograde transport from the Golgi to the ER, as well as intra-Golgi trafficking

Clathrin

A protein complex involved in the formation of vesicles for endocytosis and transport from the Golgi to the plasma membrane or lysosomes.

Cargo Receptors

Membrane proteins that bind cargo (such as proteins or lipids) and help direct them to vesicles for transport to their target compartments.

Anterograde Transport

The movement of proteins and vesicles from the ER to the Golgi apparatus and then to their final destinations, such as the plasma membrane or secretory vesicles.

Retrograde Transport

The movement of proteins and vesicles from the Golgi apparatus back to the ER or from endosomes back to the Golgi.

ER Resident Protein

A protein that is localized to the ER and is not meant to leave. These proteins typically assist with protein folding or quality control in the ER.

Golgi Resident Protein

A protein that resides in the Golgi apparatus and typically participates in post-translational modifications or sorting of proteins for transport.

KDEL Signal Sequence

A sequence of amino acids (Lys-Asp-Glu-Leu) found at the C-terminus of some ER resident proteins that signals the protein’s retention in the ER. KDEL receptors in the Golgi recognize this sequence to help return proteins to the ER.

Sar1

Formation of COPII coated vesicles requires the small G-Protein ______

Sar1-GTP

A small G-Protein that regulates COPII vesicle formation at the ER, binds to the ER membrane and recruits COPII proteins to initiate vesicle budding.

Arf1-GTP

A small G-Protein that regulates COPI vesicle formation at the Golgi and is involved in vesicle trafficking in retrograde transport, activated by GTP binding and helps recruit COPI coat proteins.

C-terminus

Which terminus is the KDEL sequence found at C-terminusR

Resident ER proteins

Which proteins have the KDEL retrieval signal at their C-terminus?