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 reactions  How 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 degradation  Endosomes: sorting of endocytosed material  Peroxisomes: 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)