1. Compartmentation of Eukaryotic Cells
a. Different intracellular compartments  distinct functions
i. Cytosol
1. Main compartment of Cytoplasm
a. Excludes membrane bound organelles
2. Gelatenous mass
a. ~20% protein by mass
3. Cytoribosomes
4. Enzymes
5. Cytoskeleton
ii. Membrane Bound organelles
1. Nucleus
a. Site of DNA & RNA synthesis
b. Envelope has a double membrane structure
c. Houses genome
2. Endoplasmic Reticulum (ER)
a. Continuous membrane
b. Synthesis of lipids and proteins
c. Ca regulation
d. Rough ER
i. Ribosomes on surface (makes it rough)
ii. Protein Synthesis
e. Smooth ER
i. No ribosomes
ii. Lipid Synthesis
iii. Ca store
3. Golgi Apparatus
a. Near nucleus
b. Modification, Synthesis and sorting of protein/lipids
made in ER
c. Vesicles on either side
4. Lysosome
a. Contains enzymes for intracellular digestion of
macromolecules and organelles
b. Garbage collector of the cell
5. Endosome
a. Contains and sorts endocytosed material
b. Can fuse with Lysosome
i. Degradation
6. Vacuole
a. Major feature of
i. Plant
ii. Fungal cells
b. Storage or Degradative Functions
7. Mitochondrion
a. Double membrane structure
b. ATP synthesis
c. 1700/cell
8. Chloroplast (plants)
a. Carbon fixation by photosynthesis
b. Double membrane structure
c. Thylakoid Membrane
d. ATP synthesis
e. CO2 fixation
9. Peroxisome
a. Oxidation of toxic molecules
b. Fatty acid breakdown
b. Relative volume of major intracellular compartments in individual cells
i. Membrane-bound organelles
1. ~50% of cytoplasm volume
2. Area and mass of intracellular membrane usually much greater
than plasma membrane (12-25x)
ii. Relative amounts of organelle types differ in different cells
1. Difference reflect functions of cells in different tissues
c. Sorting of proteins to different membrane compartments
i. Synthesis of almost all cell proteins begins in the cytosol
1. Mitochondrial proteins dont
ii. Fate of protein depends on the sorting signals found in the amino acid
sequence
1. If no sorting signal: protein stays in cytosol
2. If sorting signal present: protein goes to specific organelle
iii. Specific sorting signals could be required for retention in, or export
from, a compartment
iv. Signals located in transported vesicle or protein are recognized by
receptor protein in target organelles
v. Two types of sorting signals
1. Signal peptide (or signal sequence)
a. Continuous stretch of amino acids
i. Usually at end of polypeptide
ii. N’ end
b. May be removed by a signal peptidase
i. It is a protease
c. Experimental manipulation
i. Protein with signal sequence in ER
ii. Protein with out signal sequence in Cytosol
iii. Put signal sequence on protein in cytosol
1. Protein in cytosol moved into ER
iv. Protein with signal sequence removed moved
into cytosol
1. Signal sequence is sufficient for sorting
d. Examples
i. Cytosol to
1. ER
2. Chloroplasts
3. Mitochondria
4. Peroxisomes
5. Nucleus
ii. Golgi to ER (retention)
2. Signal patch
a. Composed of non-continuous segments of amino acid
sequence
i. Nuclear Localization Sequences
b. Identifying NLS
i. Comparing a mutated NLS to a wild type nonmutated NLS
ii. Identify difference in amino acid sequence
1. Determine which amino acids are
necessary for NLS function
c. Formation of patch requires protein folding
d. Examples
i. Nuclear import
d. Three main ways proteins move into compartments
i. Gated Transport
1. Selective gate: active transport of specific macromolecules;
small molecules diffuse freely
2. Example
a. Import through nuclear pore
ii. Transmembrane Transport
1. Use membrane-bound protein ‘translocators’
2. Transported protein unfold during transport
3. Example
a. Cytosol  mitochondria
4. Protein assisted pathway
iii. Vesicular transport
1. Membrane “sidedness’ and protein orientations are preserved
2. Example:
a. ER  Golgi
e. Methods
i. Autoradiography
1. Allows visualization of biochemical processes
2. Process
a. Radioactively labeled
b. Specimen fixed for microscopy/autoradiography
i. Dipped in liquid emulsion
1. Similar to old cameras
3. Can locate the proteins that are made up by the amino acids
that were labeled
4. Pulse Chase experiment
a. Pulse
i. Brief incubation with radioactivity during which
amino acids are incorporated into proteins
b. Chase
i. Period when the tissue is exposed to the
unlabeled medium
ii. Additional proteins synthesized using
nonradioactive amino acids
5. Secretory pathway
a. Technique first defined this pathway
ii. Green Fluorescent Protein (GFP)
1. Isolated Jellyfish gene
a. Produces GFP proteins
i. Emit green light
2. Allows to follow the dynamic movements of specific proteins in
the living organism
a. Detected by UV microscopy
iii. Subcellular Fractionation
1. Vesicles derived from different organelles have different
properties
a. Allows for separation from one another
2. Fractions identified by electron microscopy
iv. Cell-free systems
1. Isolation of a microsomal fraction by differential centrifugation
a. Able to see that isolated parts of the cell had remarkable
activities
b. Homogenate and shove thing down tube
2. RER vesicles trap newly synthesized secreted proteins
3. (Siekevitz,Palade)
v. Genetic mutants
1. Genes encode for abnormal proteins
a. Unable to carry out typical function
b. Characteristic frequency
2. Provides information on the function of the normal protein
3. Yeast secretory pathway established this way