Protein Sorting

ISAT 351, Spring 2004

College of Integrated Science and Technology

James Madison University

Intracellular Compartments and Protein Sorting

Many chemical reactions in the cell are mutually incompatible (protein synthesis and degradation)

How does the cell control these reactions? Intracellular compartments are used to segregate

and isolate different chemical reactionsHow do proteins know the correct

compartment and how are they transferred? Signal sequences direct protein traffic

Membrane -Bound Compartments

Endoplasmic reticulum (ER): synthesis and modification of lipids and proteins for distribution

Golgi apparatus: modification, sorting, and packaging of proteins for delivery

Lysosomes: intracellular degradationEndosomes: sorting of endocytosed materialPeroxisomes: oxidation of toxic molecules

Protein Sorting is in One Direction

Why is this so?•Amino acid sequence defines protein fate•Some proteins synthesized in cytosol , then transported; other proteins complete synthesis at organelle•Post-translational modification of protein

•Gradient of immature-to-mature protein may be localized in compartments

Protein Transport

Mechanisms

1. Transport through pores (nucleus)

2. Transport across membranes (chloroplast and mitochondria)

3. Transport by vesicles (ER and Golgi)

Protein Sorting Signal Sequences

Signal sequences are a continuous stretch of amino acids (15 to 60) within the protein to be sorted.

Specific sequences direct the protein to the Nu, MT, CP, peroxisomes, or ER

Cytosolic proteins lack the signal sequence

Nuclear Protein Transport

Nu proteins are synthesized in the cytosol and actively transported via Nu pores

Nuclear localization signal (+ charged sequence) unique to Nu proteins

Mitochondria Protein Transport

Nu-encoded proteins synthesized in cytosol and imported by Mt receptor

Protein unfolds during transport refolds internally

Signal sequence removed Similar mechanism for CP

Transport into the ER

Proteins enter the ER during protein synthesis ER lumen, ultimately for secretion ER membrane, ultimately for membrane

proteins

The ER signal sequence directs the ribosome to the RER

RER

Secretory Proteins are Synthesized across RER-M into the RER Lumen

Integration of Transmembrane Protein into Membrane

Post-translational Modification of Proteins in the RER

• Post-translational modifications of protein• Gradient of immature-to-mature protein may be localized

in compartments• Traffic is unidirectional, from ER to golgi

• In ER, protein is synthesized and modified• In golgi, protein is modified and sorted• Vesicle traffic (fission and fusion events) move protein,

ultimately to plasma membrane

ER Protein Glycosylation

Oligosaccharide side chains (sugars) are added to many proteins in the ER, producing glycoproteins

Functions of glycosylation: Protection from degradation Transport and packaging signals, Cell communication when displayed on the

outer membrane as glycocalyx

ER Glycosylation: Oligosaccharide Attachment

ER Glycosylation

Oligosaccharide may be further modified downstream

Transport vesicles carry glycoprotein to to golgi QC failures:

Cystic fibrosis: membrane protein improperly folded Alzheimer’s disease: improper clipping of amyloid

Transport Vesicles

Transport vesicles shuttle proteins between various organelles and to the plasma membrane (exocytosis)

Vesicles that bud from membranes have a distinct protein coat (coated vesicles) Specific marker proteins on the surface of vesicles

(SNAREs) bind to target membranes

Vesicles fuse to the target membranes and release the transported molecules

Vesicle Traffic

Golgi Apparatus

Golgi Apparatus Organization & Functions

• Stacks closest to ER (“cis” face) receive vesicles’ contents from ER

• Proteins modified (e.g., glycosylation or clipping) in subsequent cisternae

• Transport via series of fission and fusion events• Furthermost stacks (“trans” face) release vesicles that

travel to PM• Each compartment contains unique enzymes; thus,

gradient of immature to mature proteins

Transport Vesicle Docking is Mediated by Proteins

Transport Vesicle Fusion is Mediated by Proteins

Exocytosis Releases Secretory Proteins

Constitutive vs. Regulated Secretion

All cells are capable of constitutive secretion

Regulated secretion requires an extracellular stimulus Example: insulin

release

Endocytosis

Endocytosis: cells take up fluid, molecules, and other cells

Pinocytosis involves the ingestion of fluids, molecules, and small particles

Phagocytosis involves the ingestion of large particles and microorganisms

Ingested material is delivered to the lysosome

Phagocytosis

Specialized phagocytic cells (e.g.,

macrophages) can ingest invading microorganisms

Lysosome

•Lysomes contain hydrolytic enzymes that digest both intra-and extracellular materials•Enzymes are most active in acidic conditions•Not just a dump: Membrane recycling

Pathways to the Lysosome

Questions to Think About

How does compartmentalization contribute to protein sorting?

What are some consequences of misprocessing? What roles do proteins play in secretion?

Signals? Vesicle traffic?

How do membrane lipids recycle? (hint: endocytosis)