Transcript No Slide Title

Intracellular Compartments
and Protein Sorting
The Endoplastic Reticulum
Pages 727-745
3 Main Functions of the ER
1. Site of protein synthesis
-Transmembrane proteins
-Water-soluble proteins (cell exterior)
2. Site of lipid biosynthesis for most of the organelles
3. Storage of calcium in the ER lumen
EM of Rough ER
Pancreatic exocrine cell
Smooth ER
-a point where vesicles
bud from the ER in
transport to the Golgi
-Prominent in cells
which specialize in
lipid metabolism,
synthesize steroid
hormones, and in cells
involved in
detoxification
reactions
Isolation of Rough and Smooth
Microsomes from the ER
Purified Rough ER Fraction
Co- and Post-Translational
Protein Translocation
Free and
MembraneBound
Ribosomes
Co-translational
process
The Signal Hypothesis
-Signal Sequences were
first discovered in the
1970s in ER proteins
-The signal hypothesis
was tested by genetic and
biochemical experiments
Signal-Recognition Particle
-SRP guides the ER signal sequence to the ER membrane
and binds with the SRP receptor
-SRP homologues are found in all organisms that have
been studied
6 Proteins
Signal-Sequence Binding Pocket
-ER signals vary in amino acid
sequence but each has at least 8
nonpolar amino acids
-The binding pocket is a large
hydrophobic pocket lined by
methionines
-methionines can accommodate
sequences of different size and
shape
ER Protein
Targeting
-An ER Signal is
recognized twice
Ribosome bound to the Sec61 Translocator
When a protein is not transversing the pore, either a lumenal ER
protein serves as a plug or the translocator itself can close the pore
3-4 Protein Subunits creates a water-filled pore
Continuous Pore Joining of the ER
Lumen and the Ribosome
3 Methods of Protein Translocation
Chaperone Proteins prevent the
protein from folding in the cytosol
20 AA / ATP
Soluble Protein Translocation
The ER signal sequence alone is not
sufficient For signal cleavage, an adjacent
cleavage site is needed
The ER signal sequence has 2 functions:
1. it directs the protein to the ER and
2. is serves as a start-transfer signal
Single-Pass Transmembrane Protein
Hydrophobic sequence
Transmembrane Protein with an
Internal Signal Sequence, Method 1
Orientation of the start-transfer sequence
Determines which protein segment is
moved across the membrane into the ER lumen
Transmembrane Protein with an
Internal Signal Sequence, Method 2
Double-Pass Membrane Protein
All copies of the same protein will have the same
orientation in the lipid bilayer creating an asymmetrical ER membrane
Multipass Membrane Protein
Ex. Rhodopsin in rod
photoreceptor cells
ER Resident proteins
-They contain an ER retention signal of four amino acids
At their C terminus that is responsible for retaining the
protein in the ER
Return to ER signal
-Lys-Asp-Glu-Leu-COO–
Protein disulfide isomerase (PDI) catalyzes the oxidation
of free SH groups on cysteines to form S-S bonds. All
proteins in the lumen of the ER and other organelles and
the extracellular space are disulfide-bonded, while those in
the cytosol are not
N-Linked Glycosylation
-Glycoproteins – proteins containing
sugar residues
-Linked to Asparagines in the sequences
Asn-X-Ser and Asn-X-Thr, where X
cannot be Pro
-Transfer is catalyzed by oligosaccharide
transferase which has its active site on the
lumenal side of the ER
Protein
Glycosylation
The precursor oligosaccharide is
held in the ER membrane by a
long and very hydrophobic lipid
molecule, dolichol
Precursor
Oligosaccharide
Synthesis
-The sugars are first activated in the
cytosol by forming nucleotide-sugar
intermediates
-O-linked oligosaccharides
are linked to hydroxyl
groups of Ser and Thr
residues and this
occurs in the Golgi
Glycosylation in Protein Folding
Calreticulin and calnexin
both require calcium
Degradation of Misfolded ER Proteins
Retrotranslocation or dislocation
occurs via the same translocator
complex, Sec61
Unfolded Protein
Response in Yeast
GPI Anchor Attachment to Proteins
The protein can be released through cleavage by a specific phospholipase
GPI - glycosylphosphatidyl-inositol
Lipids and Membrane Components
Phosphatidylethanolamine
Phosphatidylserine
Phosphatidylcholine
Cholesterol
Ceramide (Sphingomyelin)
Glycolipids
Synthesis of Phosphatidylcholine
Occurs exclusively in the cytosol
This step enlarges the lipid bilayer
Role of Phospholipid Translocators
Phospholipid Exchange Proteins
-These are water-soluble
proteins which transfer
individual phospholipid
molecules between
membranes
-PC and PS are imported
into the Mito this way
and then some of the PS
is converted to PE