BIO 330 Cell Biology
Lecture Outline
Spring 2011
Chapters 12 & 22: The Endomembrane System and Protein Folding & Sorting
I. Overview of Endomembrane System
II. The Endoplasmic Reticulum
A. Rough vs. smooth ER
Structural and functional differences
Rough – protein folding and trafficking
Smooth –
Drug detoxification
Carbohydrate metabolism (CYP 450)
Calcium storage
Steroid biosynthesis (HMG-CoA reductase)
B. Phospholipid production
Specific phospholipid translocators create leaflet asymmetry
Phospholipid exchange proteins carry phospholipids from ER to mt and cp membranes
C. Protein synthesis and sorting (Chapter 22)
1. Brief review of translation
2. Cotranslational import – ER
Signal sequence targets a nascent polypeptide to the ER
Signal recognition particle (SRP) binds ribosome-mRNA-polypeptide to ER
SRP “pauses” translation
SRP binds at translocon
SRP receptor
Ribosome receptor
Pore protein
Signal peptidase
Translation resumes and continues simultaneously with import of polypeptide
a. Soluble protein pathway
Protein folding initiates immediately during import
BiP aids proper folding – uses ATP
Protein disulfide isomerase helps proper disulfide bonds to form
Improper or lack of folding initiates quality control responses
Unfolded protein response (UPR)
Synthesis of most proteins is reduced
ER-associated degradation (ERAD)
Proteins are degraded in cytosol by proteasomes
b. Integral membrane protein pathway
Begins similarly to soluble proteins
Stop-transfer sequence pauses translation during import
Allows folding of a-helix or other structure, which then moves
sideways out of translocon into membrane
BIO 330 Cell Biology
Lecture Outline
Spring 2011
Translation resumes until another stop-transfer sequence is
encountered, or until translation is finished
Internal start-transfer sequence allows N-term to be positioned in
cytoplasm
Membrane proteins may contain both stop-transfer and internal starttransfer sequences, allowing for any possible orientation of the
protein in the membrane
3. Proteins are trafficked to Golgi in vesicles
Proteins for export will be inside the vesicle (soluble)
Membrane proteins will be built into the membrane of the vesicle
Proteins which should remain in ER have a KDEL signal
Tells Golgi to return the protein to the ER via retrograde transport
4. Posttranslational import – mitochondria, chloroplasts, peroxisomes, nucleus
Translation is completed in cytosol, but proteins remain unfolded
Import into nucleus, mt, cp, and peroxisome use distinct but somewhat
analogous processes…details to follow
III. The Golgi Complex
A. Structure
Series of cisternae
CGN – cis-Golgi network
Medial cisternae
TGN – trans-Golgi network
B. Movement through Golgi
Two models
Stationary cisternae model
Each cistern is a stable structure
Cisternal maturation model
CGN becomes medial cisternae
Medial cisternae become TGN
Accurate model may be a merger of these two models
Anterograde vs. retrograde transport
IV. Glycosylation
Begins in ER, completed in Golgi
O-linked vs N-linked glycosylation
N-glycosylation: addition of oligosaccharide to N on asparagines residues
O-glycosylation: addition of oligosaccharide to O of hydroxyl in serine or
threonine residues
N-linked glycosylation
Core glycosylation occurs in ER
Dolichol phosphate in ER membrane
2 GlcNAc & 5 mannose sugars are added to cytosolic face
BIO 330 Cell Biology
Lecture Outline
Spring 2011
Oligosaccharide is flipped
Mannose and glucose units are added
Completed core oligosaccharide is transferred from dolichol to
asparagines residue of a polypeptide; catalyzed by
oligosaccharyl transferase
Core oligosaccharide is trimmed
Core glycoslyation occurs cotranslationally and aids proper folding
Glycoprotein moves to Golgi
Terminal glycosylation occurs throughout Golgi
Modifications to core oligosaccharide create specificity
N-linked glycosylation contributes to asymmetry
All oligosaccharides are attached to exoplasmic side of integral proteins
V. Protein Trafficking
A. ER-specific proteins
Retention tags
RXR
Retrieval tags
KDEL and KKXX
B. Lysosome-specific proteins
Mannose-6-phosphate tags
Mannose-6-phosphate receptors in membrane of TGN  endosomes  lysosomes
C. Secretory pathways
Constitutive secretion
“Default” pathway
Regulated secretion
Immature secretory vesicles develop from TGN
Maturation involves condensation +/- proteolytic processing
Vesicle waits near surface until triggered
VI. Exocytosis and Endocytosis
A. Exocytosis
Steps in exocytosis
Approach / docking
Fusion of membranes
Rupture of plasma membrane
Discharge of vesicle
Regulation by calcium
Polarized secretion
B. Endocytosis
Steps in endocytosis
BIO 330 Cell Biology
Lecture Outline
Spring 2011
Membrane invagination
Pocket pinches off
Membrane closes, forming an endocytic vesicle
Vesicle separates from plasma membrane
Most vesicles develop into endosomes  lysosomes
Receptor-mediated endocytosis
Specific internalizations
LDL internalization
Clathrin-coated vesicles
Can lead to desensitization
VII. Coated Vesicles
A. Overview of coated vesicles
Types: clathrin, COPI, COPII
Clathrin-coated vesicles transport proteins from TGN to endosomes
COPI-coated vesicles: retrograde transport from Golgi to ER
COPII-coated vesicles: anterograde transport from ER to Golgi
Caveolin-coated vesicles: shallow pits in plasma membrane
B. Assembly of coats drives formation of vesicles
E.g., Clathrin, dynamin, adaptor proteins form triskelions, which then assemble into a
cage formation
C. SNARE proteins promote fusion of vesicles and target membranes
v-SNARE: vesicle-SNAP receptors
t-SNARE: target-SNAP receptors
Rab GTPases lock t-SNAREs and v-SNAREs together
VIII. Import of Proteins into Mitochondria & Chloroplasts
IX. Import of Proteins into Nucleus