Biol 102 Unit 3

Protein Transport

Proteins destined for secretion are synthesized _______ on rough endoplasmic reticulum (ER)-associated polyribosomes and then are moved _______ via vesicles.

co-translationally, post-translationally

Jamieson and Palade Pulse chase experiment

1) pulse with radioactive amino acids

2) chase with regular amino acids

3) watch where the proteins go

To establish the path that secretory proteins take from their synthesis in the rough ER to their secretion outside the cell. Done to observe trajectory of protein movement through a cell during synthesis.

Ribosomal Subunits

a large subunit and a small subunit. These subunits come together during protein synthesis

Free Ribosomes

Ribosomes floating freely in the cytoplasm

Rough ER-associated Ribosomes

Ribosomes attached to the cytoplasmic surface of the rough ER. These ribosomes synthesize proteins destined for secretion

Common Pool

Both free and rough ER-associated ribosomes use the same pool of ribosomal subunits.

Proteins that are transported to the nucleus contain

Nuclear Localization sequence

Proteins destined for the nucleus are transported into the nucleus via the

Nuclear Pore Complex

LaminsProteins that binds to the nuclear localization sequence

Proteins that line the inner nuclear membrane to give structure to the nucleus

Import Receptors

Proteins that binds to the nuclear localization sequence

Proteins that binds to the nuclear import receptor in the nucleus

Ran - GTP

Nuleoporins

Proteins in the nuclear pore with FG-repeats

Sectory protein transport

1) MRNA recognizes the signal

2)MRna ceases translation

3)Signal regcognition partical attaches
4)signal recongisiton particle receptor attaches and postions protien at translocoon(hydrolysinig gtp)

5)Singla pepidase cleaves NLS and protein goes into the nucleaus

Where is the signal sequence

on the N-terminal of the poly peptide

Exocytosis

process by which cells transport substances from inside the cell to the outside by vesicles. Vesicles containing the material fuse with the plasma membrane, releasing their contents to the extracellular space.

Signal Recognition Particle

A protein-RNA complex that helps guide newly made proteins to the ER.

Signal Sequence:

A short peptide that directs a protein to the ER for further processing.

exocyotic pathway steps

1) Proteins synthesis in the ER

2) Transport to the goli in er vesicle

3) tagged for destination

4) secreted

Signal Recognition Particle Receptor (SRPR)

A protein on the ER membrane that helps the SRP bring proteins to the ER.

Signal Peptidase:

An enzyme that removes the signal sequence from proteins once they are in the ER.

parts of the endomembrane system

Nuclear Envelope

Endoplasmic Reticulum

Golgi Apparatus

Lysosomes

Vesicles

Plasma Membrane

Steps of Cell Fractionation

1)Homogenization

2)Differential Centrifugation

• low-speed spin: Sediments nuclei and cell debris.

• Medium-speed spin: Sediments mitochondria, lysosomes, and peroxisomes.

• High-speed spin: Sediments microsomes (fragments of ER) and small vesicles.

• Very high-speed spin: Sediments ribosomes and macromolecules.

Autoradiography

A technique that uses radioactive isotopes to visualize the distribution of proteins or nucleic acids in a sample. It allows researchers to track and analyze molecular interactions and labeled cells.

Protein Sorting Pathway

Begins in the cytosol where proteins are intiially synthesized, and then finish in either the cytosol or the ER. If signal peptide sequence is present the protein is sent through the endomembrane system starting with the ER.

Regulated versus constitutive Exocytosis

Regulated exocytosis occurs in response to specific signals, while constitutive exocytosis is a continuous and unregulated process that delivers materials to the cell surface.

Steps for preparing a cell-free system

• Lyse the cell - break open the cell to release its contents

• Fractionation - isolate rough ER (ER microsomes) using differential centrifugation

• Reconstitution - Add protein synthesis machinery to enable translation.

Isolation of stripped Microsomes

Extract rough microsomes and and treat with high salt and puromycin. Results in a stripped microsomes with nascent protein chain insertion into the ER lumen for use in-vitro. Puromycin inhibits protein synthesis.

Detached Polysome Extraction

treat rough microsomes with non-ionic detergent. Yields functional ribosome with nascent protein chain attached.

Protease Protection Assay

Add protease to a sample of cells to observe digestion of a protein. Done to determine if newly synthesized secretory proteins are inserted into RER Co or post-translationally into the RER lumen, revealing their localization and confirming their translocation during synthesis.

Ionic vs. Nonionic detergent

refers to two classes of detergents used to either solubilize membrane proteins or maintain protein structure during extraction processes. Ionic detergents disrupt ionic bonds, while nonionic detergents stabilize proteins.