Protein Targeting and Transport

Protein Localization Overview

• Membrane-enclosed organelles have specific locations within the cytosol.
• Protein localization determines protein functions within cells.

Protein Transport Methods

• Proteins are transported via four primary mechanisms:
  • Protein translocation: Protein translocators move proteins across membranes (e.g., from the cytosol to the ER lumen, mitochondria, or ER).
  • Gated transport: Selective gates control the movement of proteins (e.g., nuclear transport).
  • Vesicular transport: Transport vesicles are used to move cargo
  • Engulfment/autophagy: Which involves formation of compartments.

Sorting Signals

• Proteins are sorted using two main types of signals:
  • Signal sequences: Continuous amino acid stretches (typically 15-60 residues) direct proteins to specific locations like the ER, mitochondria, chloroplasts, peroxisomes, or nucleus. They are recognized by sorting receptors.
  • Signal patches: 3D arrangements of amino acids which are brought together when the protein folds.

Nuclear Envelope and Transport

• The nuclear envelope encloses the DNA and defines the nuclear compartment.
• It consists of two membranes with distinct functions. -The inner nuclear membrane has proteins that bind chromatin and the nuclear lamina.
• The space between inner and outer membranes of the nucleus is continuous with the ER lumen.
• Traffic happens constantly between the cytosol and the nucleus
• The nuclear lamina, made of nuclear lamins, provides structural support inside the nucleus and Anchors chromosomes and NPC's.

Nuclear Pore Complexes (NPCs)

• The nuclear envelope is perforated with NPCs which serve as core complexes.
• These are composed of over 30 nucleoporin proteins.
• Each pore complex contains aqueous channels where small, water-soluble molecules passively travel

Nuclear Localization Signals (NLS)

• Signal sequences or patches work to direct proteins to the nucleus
• They are typically rich in lysine and arginine amino acids.

Nuclear Import Receptors

• Soluble cytosolic proteins bind to both the nuclear localization signal and nucleoporins.
• Most signals are recognized by nuclear import receptors (karyopherins) to start nuclear import.

Ran GTPases

• R GTPases act as molecular switches and are required for the translocation of RNA and proteins through the NPC.
• GTPase-activating proteins (GAPs) trigger GTP hydrolysis to convert Ran-GTP to Ran-GDP.
• Guanine nucleotide exchange factors (GEFs) promote GDP exchange for GTP to convert Ran-GDP into Ran-GTP.

Regulation of Nuclear Transport

• Nuclear localization and export signals can be turned on or off by phosphorylation.

Fluorescent Labeling

• Fluorescein isothiocyanate (FITC) labels proteins, antibodies, peptides, hormones, and other amine-containing molecules using a green fluorescent dye.

Transport into Mitochondria

• Proteins destined for mitochondria are synthesized as precursor proteins in the cytosol and then translocated post-translationally.
• Mitochondrial precursor proteins typically have a signal sequence at the N-terminus, which is rapidly removed following import.

Mitochondrial Translocators

• TOM complex functions across the outer mitochondrial membrane.
• TIM23 and TIM22 complexes work across the inner membrane.
• The OXA complex mediates the insertion of inner membrane proteins made within the mitochondria.

TOM Complex Requirements

• Necessary in order to import nucleus-encoded mitochondrial proteins.
• Transports signal sequences into the intermembrane space and helps to insert transmembrane proteins into membrane.

TIM23 Role

• moves proteins into the matrix space along with helping embed transmembrane proteins into membrane

TIM22 Role

• Mediates inner membrane proteins, like carrier protein that moves ADP, ATP and phosphate

Signal Peptidases

• These remove N-terminal signal sequences.

Mitochondrial Electron Transport

• This transfers H across inner membrane to create a gradient.

Transport to Chloroplasts

• Protein translocation closely resembles transport into mitochondria.

Chloroplast Structure

• Chloroplasts have stroma, lamellae, and thylakoids.

Thylakoid Transport

• This requires special thylakoid signal sequence

Transport Summary

• Transport involves recognition, translocation, transport, cleavage, and folding.

Peroxisomes

• Also known as microbodies, they contain oxidative enzymes like catalase and urate oxidase.
• They differ from Lysosomes in the type of enzymes that they hold
• They are surrounded by a single membrane, and they lack DNA and ribosomes.
• All proteins must be imported.

How Peroxisomes Oxidize

• Molecular oxygen is used to remove hydrogen atoms from organic substrates and create hydrogen peroxide (H2O2).
• Catalase converts hydrogen peroxide into water and oxygen.

Peroxisome Role

• They perform beta oxidation of long chain fats, yeast and plan cells
• They are used in the break down of very long fatty acids

Peroxisome Function

• They catalyze formation reactions of plasmalogens which are a class of phospholipids in myelin.
• Plasmalogen deficiencies result in abnormal myelination and neurological disorders.

Diseases

• Zellweger syndrome involves empty peroxisomes with abnormalities in the brain, liver, and kidneys due to mutations.

Peroxisome Import

• Import resembles nuclear transport via receptors.
• Proteins do not have to be unfolded to be transported into peroxisomes.

Endoplasmic Reticulum (ER)

• Proteins can be transported here
• Rough ER (RER) has ribosome-covered surface for protein synthesis.
• Smooth ER (SER) lacks ribosomes and is involved in lipid synthesis.
• ER Abundance: ER abundance depends on cell specialization.

Functions of the RER

• Site of protein synthesis
• Involved in glycoprotein formation
• Produces precursors of lysosomes

Functions of the SER

• Lipid synthesis occurs here.
• Hepatocytes uses smooth ER to create particles
• Enzymes used to detoxify substances

Importance of Cytochrome P450

• In the smooth ER of the liver, specific enzymes detoxify and convert water-insoluble drugs or metabolites into water-soluble substance.

ER-bound Polyribosomes

• These target the ER with proteins that have an ER signal sequence
• These contain proteins for the cytosol and cytoskeleton
• A Signal-Recognition Particle (SRP) recognizes the signal from the sequence

Sec61 Complex

• It acts as a protein translocator.
• It allows for proofreading.
• Is gated, which mean it can be opened and closed.

How Localization Happens

• This either can happen while proteins are being translated (co-translational) or after the protein has already been translated (post-translational)

Internal ER Sequences

• Some function as stop transfer peptides to anchor to ER membrane

Oligosaccharides and Protein Folding

• These work as tags for proteins in ER
• calnexin is a carbohydrate bidning protein.

ER Protein Export

• If misfolded, exported and degraded