Protein Targeting

What synthesizes proteins and where does it take place

Ribosomes and in the cytosol

Where can proteins be targeted to

• They can exit the cell

• Integrate into membranes

• Enter subcellular compartments

What are signal sequences and what do they do

Short amino acid motifs that serve as addresses guiding proteins to specific locations

Why does protein targeting occur exclusively in eukaryotic cells

It allows for subcellular organization which aids in compartmentalizing metabolic pathways

What signal sequence targets ribosomes synthesizing proteins and directs them into the ER

N-terminal signal sequences

Features of N-terminal signal sequences

• 13-20 amino acids in length

• Contains about 10-15 hydrophobic amino acids

• Includes one or more positively charged amino acids near the N-term

• Has a polar short sequence near the C-term

• Contains a cleavage site

Signal Recognition Particle (SRP)

A conserved ribonucleoprotein complex that directs specific proteins into the ER by binding to signal sequences of nascent polypeptides

Nascent polypeptide

A newly synthesized amino acid chain that emerged from the ribosome

How does the SRP pathway work

1. The SRP recognizes and binds to a hydrophobic N-terminal signal sequence from a nascent polypeptide

2. Upon binding, the SRP’s Alu domain interacts with the ribosomal GTPase center, pausing translation, preventing protein misfolding or aggregation in the cytosol

3. The SRP- Ribosome-Nascent Chain (RNC) complex moves to the ER membrane, where the SRP binds to the SRP receptor SR (GTPases)

4. The RNC is transferred from the SRP/SR complex to the Sec61 translocon

5. The SRP and SR dissociate following GTP hydrolysis. Translation resumes and the growing polypeptide is threaded directly into the ER lumen or integrated into the ER membrane

What is the overall goal of the SRP pathway

To translocate proteins to the ER co-translationally

Where do proteins undergo post-translational modifications and list an example

In the ER and Glycosylation

What happens to proteins after undergoing post-translational modifications

They get packaged into transport vesicles and are taken to the Golgi apparatus to get sorted

How does the Biosynthetic / Secretory pathway work

1. In the ER: Ribosomes on the rough ER synthesize proteins targeted for secretion or membrane insertion

2. ER Exit Sites (ERES) & ERGIC: Proteins destined for transport are captured into COPII-coated vesicles. These vesicles leave the ER at specialized sites (ERES) and travel to the ER-to-Golgi intermediate compartment (ERGIC)

3. Golgi Complex: Vesicles from the ER fuse to form the cis-Golgi , where proteins are further processed, sorted, and packages, and move through the Golgi cisternae toward the Golgi network (TGN)

4. Post-Golgi Transport (TGN): The TGN acts as a sorting hub, packaing proteins into specific vesicles directed to their final destinations: lysosomes, the plasma membrane, or extracellular space

What is the main goal of the biosynthetic secretory pathway

To synthesize, modify, and transport proteins / lipids to the plasma membrane, lysosomes, or extracellular space by moving cargo through the ER and Golgi in an anterograde manner

Glycoprotein

A linkage between an oligosaccharide to a side group on a peptide

N-linked glycosylation

Occurs on the nitrogen atom of Asparagine (Asn) or Arginine (Arg) side chains

O-linked glycosylation

Occurs on the oxygen atom of Serine (Ser), Threonine (Thr), and Tyrosine (Tyr)

Core oligosaccharides are built on dolichol phosphate by the successive addition of what sugar units

monosaccharides

Where do oligosaccharides get transferred to and within what

Asn residues in the ER lumen

Tunicamycin function

To block step 1, the initial step of N-linked glycosylation

Where can O-linked glycosylation take place

In the Golgi or cytosol

Golgi Apparatus function

Responsible for modifying, sorting, and packaging proteins and lipids from the Endoplasmic Reticulum (ER) into vesicles for delivery to specific destinations

Why will a protein get directed to the plasma membrane

For incorporation as membrane proteins or secretion

Lysosome function

They break down waste materials, foreign invaders (bacteria/viruses), old cell parts (autophagy), and facilitate programmed cell death (apoptosis) and repair the plasma membrane.

Where do proteins destined for the mitochondria and chloroplasts bind chaperone proteins

In the cytosol, they are then delivered to receptors on the exterior of the organelle

Chaperone protein function

To maintain proteins in an unfolded state suitable for import

What do proteins targeted to the nucleus carry

A nuclear localization sequence (NLS)

Characteristics of a nuclear localization sequence (NLS)

• 4-8 amino acids long

• Contains several consecutive basic residues (Arg, Lys)

• Not removed after import

• Can be located anywhere within the protein sequence

Targeting to the nucleus mechanism step 1

A protein with an appropriate NLS is bound by a complex of importin α and β

Targeting to the nucleus mechanism step 2

The importin (α and β)-protein complex binds to a nuclear pore and translocates into the nucleus

Targeting to the nucleus mechanism step 3

Inside the nucleus, dissociation of importin β is promoted by the binding of Ran-GTP

Targeting to the nucleus mechanism step 4

(nucleus)- Importin α binds to Ran-GTP and CAS (cellular apoptosis susceptibility protein), releasing the nuclear protein

Targeting to the nucleus mechanism step 5

(nucleus)- Importin α and β and CAS are transported out of the nucleus and recycled. Ran is transported back into the nucleus