Intracellular Compartments and Protein

Sorting-Part 2

03.22.2023
Myungin Baek
Chapter 12
Molecular Biology
of the Cell
Sixth Edition
 The compartmentalization of Cells
The transport of molecules between nucleus and the
cytosol
The transport of proteins into mitochondria and
chloroplasts
Peroxisomes

The endoplasmic reticulum

Introduction
 Introduction

• Peroxisomes are different from mitochondria and chloroplasts

‒ Surrounded by a single membrane
‒ No DNA or Ribosomes, meaning all protein should be encoded in the nucleus
• Peroxisomes contain oxidative enzymes at a high concentration
e.g., Catalase, Urate Oxidase
 Peroxisomes Use Molecular Oxygen and Hydrogen
Peroxide to Perform Oxidation Reactions
• In peroxisome, Hydrogen peroxide is generated by using oxygen molecules

RH2+O2 -> R+H2O2

• Catalase uses H2O2 to oxidize diverse substrates (e.g., alcohol)

H2O2+R’H2-> R’+2H2O
• Peroxisomes breakdown fatty acid to acetyl-CoA through β oxidation
- mitochondria and peroxisomes perform this in mammals but only in
the peroxisomes in plant and yeast
• Peroxisomes catalyze the first reaction for forming plasmalogen (phospholipid in myelin)
- due to this function many peroxisomal disorders lead to neurological disease
 Peroxisomes Use Molecular Oxygen and Hydrogen
Peroxide to Perform Oxidation Reactions
• Peroxisomes are important in plants
• Two types of peroxisomes are well studied in plants
‒ Peroxisomes in leaves participate in photorespiration
‒ Peroxisomes (glyoxysomes) in the germinating seeds convert fatty acids into sugars
through glyoxylate cycle
 A Short Signal Sequence Directs the Import
of Proteins into Peroxisomes
• Signal sequence: an amino acid sequence (Ser-Lys-Leu) in the C-terminus
‒ Soluble receptor proteins recognize the sequence in the cytosol
‒ At least 6 different peroxins form a protein translocator
‒ Oligomeric proteins can be transported without unfolding
ü The pore is dynamic to adapt in size to the cargo molecule
‒ Soluble receptor, Pex5 recognizes the signal sequence and
enter the peroxisome and cycles back to the cytosol
Pex1 and Pex6 help the release of Pex5 to the cytosol using ATP energy
• New peroxisomes could be formed by fission of preexisting peroxisomes or
formed from the specialize peroxisomal precursor vesicles originated from ER membrane
 The compartmentalization of Cells
The transport of molecules between nucleus and the
cytosol
The transport of proteins into mitochondria and
chloroplasts
Peroxisomes

The endoplasmic reticulum

Introduction
 Introduction

• ER is organized into a netlike structure of branching tubules and flattened sacs

• ER membrane is continuous with nuclear membrane
• ER plays a critical role in lipid and protein synthesis and stores Ca2+
‒ All the transmembrane proteins and lipid for most of the organelles are produced in
the ER membrane
‒ Almost all of the proteins secreted to the cell exterior are initially delivered to the ER
lumen
 The ER Is Structurally and Functionally Diverse

• Distinct regions of the ER become highly specialized

‒ e.g., rough ER, smooth ER
• Most translocation into ER is co-translational process
‒ Translocation to mitochondria, chloroplast, peroxisomes is post-translational process
 The ER Is Structurally and Functionally Diverse

• Transitional ER is the smooth ER where transport vesicles containing proteins and

lipids bud off for transport to the Golgi

• In certain cells, smooth ER has additional functions

‒ Cells specialized in lipid metabolism (e.g., synthesize steroid hormone from
cholesterol; cells in testis) have smooth ER containing enzymes that synthesize
cholesterol and modify it to form hormone
‒ hepatocyte smooth ER is the principal site for the production of lipoprotein
particles carrying lipid in the bloodstream

• ER sequesters Ca2+ from the cytosol and releases into the cytosol
‒ Ca2+ pump transport Ca2+ from the cytosol into the ER lumen
‒ High levels of Ca2+ binding proteins in the ER allows the Ca2+ storage
‒ Muscle cells have a modified smooth ER, sarcoplasmic reticulum where Ca2+
release and reuptake occurs during muscle contraction and relaxation
 The ER Is Structurally and Functionally Diverse

• Function and Biochemistry of the ER can be studied using microsomes, a small vesicles
(~100-200μm in diameter)
‒ Smooth microsomes and rough microsomes could be separated using equilibrium
centrifugation
‒ Smooth microsomes in general are not purely from smooth ER with the exception
of
liver smooth ER and muscle sarcoplasmic reticulum
 Signal Sequences Were First Discovered in
Proteins Imported into the Rough ER
• Two types of proteins are targeted to ER
‒ Transmembrane proteins: embedded in the ER membrane; mostly destined to the
plasma membrane or membrane of other organelles
‒ Water-soluble proteins: released into the ER lumen; destined for secretion or
residence in ER lumen or another organelle
• ER signal sequences direct proteins to the ER membrane and initiate the translocation
• Signal sequence was first discovered in the 1970s in secreted proteins
‒ mRNA encoding a secreted protein was translated by ribosomes in vitro
‒ w/o microsome, the translated product became larger than secreted protein
‒ In the presence of rough microsome, the translated product became similar
size to the secreted protein
 A Signal-Recognition Particle (SRP) Directs the ER Signal
Sequence to a Specific Receptor in the Rough ER Membrane
• ER signal sequence is guided to the ER membrane at least 2 components
‒ SRP (signal-recognition particle): binds to signal sequence; recycles between ER
membrane and cytosol
‒ SRP receptor: located in the ER membrane
• ER signal sequences contain mostly 8 or more nonpolar amino acids in the center
‒ signal-sequence binding site in the SRP contains a large hydrophobic
pocket which provide flexibility to accommodate different sequences, sizes,
and shapes of signal sequence
• SRP has a rod-like structure
‒ One end binds to the signal sequence while the other end blocks the
elongation factor binding site in the ribosome
- as soon as the signal sequence emerges from ribosome,
the protein synthesis halts with allowing enough time
for the ribosome to bind to the ER membrane
 A Signal-Recognition Particle (SRP) Directs the ER Signal
Sequence to a Specific Receptor in the Rough ER Membrane
• Once SRP binds to signal sequence, it exposes a binding site for the SRP receptor
• The binding of SRP to its receptor brings the complex to the protein translocator
• SRP and SRP receptor are released and translocator transfers the polypeptide across
the membrane
 A Signal-Recognition Particle (SRP) Directs the ER Signal
Sequence to a Specific Receptor in the Rough ER Membrane
• Free ribosomes and membrane bound ribosomes are the same
‒ membrane bound ribosome synthesize the one translocated to the ER
‒ Free ribosome synthesize all the other protein encoded in the nuclear genome
• Many ribosomes can bind to the single mRNA (polyribosome)
 The Polypeptide Chain Passes Through an
Aqueous Channel in the Translocator
• The core of the translocator, Sec61 complex consists of three subunits (αβγ)
‒ α helices from the largest subunit form the channel through which the peptide passes
‒ A small α helix function as a plug which is transiently removed only when the peptide
is translocated
any advantage?
‒ The pore can also open along the seam on its side, which allows lateral translocation
to the membrane
 Translocation Across the ER Membrane Does Not
Always Require Ongoing Polypeptide Chain Elongation
• Exceptions in the co-translational translocation to the ER
‒ Post-translational translocation is common across the ER membrane of the yeast
‒ ER translocator requires accessory proteins
ü In Eukaryotes, Sec62,63,71,72 complex recruits BiP(binding protein, chaperon) to the
translocating polypeptide; ATP dependent BiP binding and release pull the protein
to the lumen
ü In bacteria, ATP dependent conformational change of the SecA ATPase
assist the peptide translocation
 In Single-Pass Transmembrane Proteins, a Single Internal ER Signal
Sequence Remains in the Lipid Bilayer as a Membrane-spanning a Helix
• After a sufficient length of polypeptide is formed, signal sequence bind to a specific
site inside of the pore, which open the pore (function as a start-transfer signal)
‒ ER Signal sequence is recog nized twice; by SRP in the cyto sol and b y a b inding site
in the pore of the translocator
‒ Signal sequence binds to both Sec61 complex and with the hydrophobic core of the
lipid bilayer through the lateral seam
ü Once released from the pore, signal sequence gets rapidly degraded
• Translocator is gated in two directions
‒ Hydrophilic portion of the protein cross the lipid bilayer through its pore
‒ Hydrophobic portion into the lipid bilayer through its lateral seam
ü Lateral gating is essential step for the integration of the transmembrane proteins
 In Single-Pass Transmembrane Proteins, a Single Internal ER Signal
Sequence Remains in the Lipid Bilayer as a Membrane-spanning a Helix
• Three ways of single-pass transmembrane proteins get inserted into the ER membrane
‒ N-terminal signal sequence initiates translocation; an hydrophobic segment in the
polypeptide stops the transfer process (stop-transfer signal); the stop-transfer signal
anchors the protein in the membrane after the signal sequence is released;

‒ In the other two cases, the signal sequence is internal

ü SRP binds to the internal signal sequence by recognizing its hydrophobic α helical
feature
ü ER signal sequence functions as a start-transfer signal
ü After release from the translocator, the signal sequence remains in the lipid bilayer
ü Internal signal sequence binds to the translocator in either of two orientations,
depending on the nearby charged amino acid; more positively charged amino acid
preceding the signal sequence (N terminal in the cytosol) vs. following the signal
sequence (N terminal in the ER lumen)
 Combinations of Start-Transfer and Stop-Transfer Signals
Determine the Topology of Multipass Transmembrane Proteins

• In multipass transmembrane proteins, polypeptide passes the lipid bilayer as hydrophobic

α helices
‒ Internal signal sequence functions as a start-transfer signal
‒ For double pass transmembrane proteins, once the translocator encounters the stop-
transfer signal the peptide is released into the lipid bilayer
 Combinations of Start-Transfer and Stop-Transfer Signals
Determine the Topology of Multipass Transmembrane Proteins
• In a more complex multipass transmembrane proteins, a second start-transfer sequence
reinitiates translocation until the stop-transfer sequence is encountered
‒ Distinction between the start-transfer and stop-transfer sequences are
mostly determined by their relative order in the growing polypeptide
 ER Tail-anchored Proteins Are Integrated into
the ER Membrane by a Special Mechanism
• Many functionally important proteins anchored in the ER membrane by a C-terminal
transmembrane α helix (ER tail-anchored proteins)
‒ Due to the location of the signal sequence in the C-terminus, SRP doesn’t have a
change to bind to it
‒ The polypeptide released from the ribosome and gets anchored to the ER membrane
through specialized targeting machinery that uses ATP (Get1, 2, 3)
 Translocated Polypeptide Chains Fold and
Assemble in the Lumen of the Rough ER
• Proteins translocated into the ER lumen are translocated to other destination or reside in
the ER lumen (ER resident proteins)
‒ ER resident proteins contains ER retention signal of 4 amino acids at their C-terminus
‒ Some of these proteins function as a catalysts that helps the ER lumen proteins to fold
and assemble correctly
ü Protein disulfide isomerase (PDI) catalyzes the disulfide bonding (S-S) between
cysteine amino acids
ü BiP pulls proteins post-translationally through Sec61; BiP binds to the incorrectly
folded proteins and prevents the proteins from aggregation
 Most Proteins Synthesized in the Rough ER Are Glycosylated
by the Addition of a Common N-Linked Oligosaccharide

• Addition of oligosaccharide to protein is the one of the major function of ER

‒ About 50% of soluble and membrane p rotein pro cessed in the ER are glyco proteins
‒ Precursor oligosaccharide (N-acetylglucosamine-mannose-glucose)is
transferred to the side chain NH2 group of asparagine
ü Membrane-bound enzyme complex (oligosaccharyl transferase) catalyzes this process
ü Dolichol (lipid) anchors the precursor oligosaccharide in the ER membrane
ü Immediately after the target asparagine reaches the ER lumen during translocation,
precursor oligosaccharide is transferred to the target amino acid
 Oligosaccharides Are Used as Tags to Mark
the State of Protein Folding
• N-linked glycosylation plays a role in protein folding
‒ ER Chaperones, calnexin (membrane bound) and calreticulin (soluble) are carbohydrate-
binding proteins that bind to oligosaccharides on incompletely folded proteins and retain
them in the ER lumen
üCalnexin and calreticulin prevent incompletely folded proteins from forming irreversible
aggregation
üCalnexin and calreticulin recognize N-linked oligosaccharides that contain a single
terminal glucose; once the terminal glucose is removed, the protein can leave the ER
üGlycosyl transferase add a glucose to the oligosaccharide of the unfolded protein that
has lost its last glucose
üUnfolded protein undergoes cycles of glucose trimming and addition until it
is fully folded
 Improperly Folded Proteins Are Exported from
the ER and Degraded in the Cytosol

• Translocated proteins that fail to fold properly, exported back to the cytosol and undergo
degradation in proteasomes (Retro-translocation)
‒ Chaperones and energy are required for maintaining unfolded state during translocation
• N-linked oligosaccharide helps to distinguish misfolded protein from folding intermediates
‒ N-linked oligosaccharide serves as a timer
ü Oligosaccharide trimmed in specific mannose (slow trimming by mannosidase) is
recognized by retro-translocation apparatus
ü Proteins that fold and exit from the ER escape from the mannosidase
 Misfolded Proteins in the ER Activate an
Unfolded Protein Response
• Cells monitor the amount of misfolded protein in various compartments
‒ Unfolded protein response is triggered by accumulation of misfolded protein in the ER
ü Increase in expression of proteins involved in retrotranslocation and protein
degradation in the cytosol, ER chaperon, etc.
‒ Misfolded proteins in the ER signal to the nucleus in three parallel pathways
ü Activation of IRE1 leads to the splicing of specific TF mRNA in the cytosol
(increased chaperone expression)
ü Activated PERK inhibits translation initiation except the one involved in UPR
ü ATF6 in ER membrane is transported to the Golgi where it is activated by protease;
ATF6 in the nucleus activates transcription of genes involved in UPR
 Some Membrane Proteins Acquire a Covalently
Attached Glycosylphosphatidylinositol (GPI) Anchor
• In the ER, glycosylphosphatidylinositol (GPI) anchor is attached to the C-terminus of
some membrane proteins destined to the plasma membrane
‒ Transmembrane segment of the protein is cleaved off during the linkage formation
‒ GPI anchored protein could be released from the plasma membrane by phospholipase
that is activated by signals
‒ GPI anchor may assist proteins to be located to lipid rafts, which segregate
the protein from other membrane proteins