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Endocytosis Pathway is mediated by CCV
CCVs bud off from the PM. The endocytic vesicles are targeted and fused to the early
endosome. CCVs can also move from the early endosome back to the PM to recycle PM
receptors; additionally, there is also cycling between the trans Golgi, PM, and early
endosomes.
MVBs contain cytosolic vesicles in its lumen
Buds from the early endosomes can go through microtubule-mediated transports to form
multivesicular bodies (MVB). The MVBs mature into late endosomes, and then
endolysosomes, and finally lysosomes. Formation of MVB is irreversible (can’t go back
to endosomes); the membrane of the MVB invaginates, pinches off, and forms the MVB
in such a way that the lumenal contents also include vesicles with cytosolic contents
inside of them. Then, MVB fuses with vesicles containing proteases and lipases, which
digest the cytosolic vesicles inside the MVB to transform it into a Lysosome.
Receptor Recycling loses ligands
Plasma membrane receptors have extracellular ligands. These extracellular ligands (such
as LDL, low density lipoprotein, which is a cholesterol carrier) can be internalized with
TM receptors in CCV vesicles, and targeted into the endosome. Since sorting of proteins
occur at the early endosome, and pH of the early endosome is lower than that of
extracellular space, some ligands separate from their receptors. The empty receptors are
recycled back to the PM by vesicles; on the other hand, the ligands are forwarded to the
lyosomes to be degraded into their constituents.
Endosome Pathway
Some TM receptors are marked for endocytosis by modification with mono-ubiquitin
(NOT polyubiquitin chains—so no proteasomes) at the PM by specialized E3 ligases.
They are subsequently digested in lysosome. Monoubiquitin-tagged receptors are
gathered into MVBs.
The mechanism is as follows:
1. At the endosome, a series of ESCRT protein complexes bind to monoubiquitinated receptors and PI(3)P
2. ESCRT complexes then pinch off the membrane towards the lumen (opposite
direction to transport vesicle budding)—note that this is not based on SNAREs or
Coat proteins!
a. First ESCRT to bind is ESCRT-0, which gathers monoubiquitinated cargo
(TM receptor proteins)
i. At this step, Clathrin might be involved, but it does not form any
cages
b. Then, ESCRT I & II recruit ESCRT-III, which forms oligomers and
deforms the membrane inwards, and then finally pinching the membrane
off as they attract each other
c. Meanwhile, the DUBs remove ubiquitin
d. When pinching off is finished, a Vps4 (AAA—remember, pulling apart!)
recycles ESCRT-III
3. MVBs (or late endosomes) now contain vesicles and cytosolic contents and fuse
with lysosomes
a. This is the final site of protein degradation in the secretory pathway, and it
degrades all secretory proteins that exit from the ER
i. The first site of possible degradation is through ERAD, in
proteasomes
ii. The only other place is here, in lysosomes; otherwise, the protein
cannot be degraded
b. Lysosomes contain enzymes that digest a variety of cellular material in
addition to proteins, as its lumen is highly acidic
i. Acidity of interior is maintained by ATP-dependent proton pumps
ii. Actually, the degradation enzymes inside the lysosome are most
active at the low pH (ex: nucleases, proteases, glycosidases, etc.)
1. This is actually regulatory, as the enzymes are kept inactive
until they reach the lysosome (it would be catastrophic
otherwise, having free nucleases flying all over the cell)
iii. Proteasomes are for proteins only!
4. Final breakdown products (smallest units possible) are transported into the cyotosl
====END OF THE SECRETORY PATHWAY ====
Nuclear Membranes are continuous with the inner and outer membranes of the ER
The perinuclear space (between inner & outer membranes) of the nuclear envelope is
continuous with the ER lumen; however, there is lateral separation between the smooth
ER, rough ER, as well as the nuclear membrane. The nucleus interior (nucleoplasm) is
continuous with the cytosol. Connections between the nuleoplasm and the cytosol are
through the nuclear pore complex; in fact, mRNA-protein complexes are exported and
protein complexes are imported/exported through these pores.
The difference between nucleoplasm import-export and ER import-export is that large
protein complexes can be transported without being unfolded and not in vesicles—even
proteasomes and ribosomes, while the ER insists on pulling things into straight chains
through the translocon pores.
The nuclear pore complex is a very large structure that forms pores through the envelope
Multiple protein subunits (nucleoporins) form the ring of the nuclear pore complex.
Additionally, long protein filaments extend into the cytosol, and also forming a basketlike structure on the nuclear side. Because the nuclear pore complex is not sealed, small
folded proteins can diffuse through directly. On the other hand, large proteins (>40kDa)
and mRNA complexes are selectively transported—but still possible.
Nuclear Targeting Signals consist of Nuclear Localization Signals (NLS) and Nuclear
Export Signals (NES)
Nuclear Localization signals are usually 1 or 2 clusters of 4-5 positively charged residues
(Lys, Arg), which are recognized by importin-α, an adaptor onto main transporter
importin-β. Other, non-classical NLS are recognized directly by importin-β. Importins are
soluble receptor proteins of α and β types. Importin α binds NLS by recognizing positive
charges and contain a separate importin-β binding domain. On the other hand, importin- β
has a binding site that is under constant competition between importin- α and Ran
GTPase (a regulatory factor controlled by GTP-switching, and related to Rab, Sar1, and
Arf). Importin-β interacts with nuclear pore (through FG-Nucleoporins) to mediate
import. Mechanism is as:
1. Importin-α/β heterodimer recognizes an NLS in the cytosol and complexes as
cargo- α- β
2. The complex transiently contacts pore during import, which allows the
complex to proceed into the nucleus
3. Once inside the nuclease, Ran GTPase displaces importin-α from the complex,
which releases the cargo from importin
a. The Ran GTPase is still in its GTP-bound state!
FG-Nucleoporins line the inside of the nuclear pore. They are proteins with long Phe-Gly
repeat sequences. Their repeats are unstructured and partially hydrophobic, and due to
their hydrophobicity, they form a flexible diffusion barrier (like a curtain). Importins and
Ran can transiently bind the FG repeats to bring cargo through. There are several
hypothesis of Nucleoporin function, all of which involve direct interactions between FGs
and importins:
1. Barrier
a. Interactions between FG repeats form a barrier (brushes or gel?)
b. Importin binding displaces the polypeptide strands, and allows the
importin the slip through
2. Binding
a. FG repeats provide multiple weak binding ites for importins
b. Importins randomly bind and re-bind
c. The high local concentration of importins around the pore increases
probability that they will move through
Nuclear Export Signals are usually 4-5 hydrophobic residues alternating with polar
residues. They are recognized by exportins (ex: CRM1). The mechanism for exportins
parallel that of the importins. Exportin and Ran-GTP bind to NES, which interact with
pore for export. Ran hydrolyses GTP to GDP and the cargo is released from exportin.
An mRNA can have more than one NLS or NES!
Ran GTPase determines the direction of nuclear transport
GTPase Activating Protein (GAP) stimulates Ran to hydrolyze GTP when it enters
cytosol. On the other hand, GDP Exchange Factor (GEF) causes Ran to release GDP and
bind GTP when it enters nucleus. Therefore, all Ran in the nucleus is GTP-bound (so that
it can displace importin- α), while most Ran outside the nucleus should be in their GDP
bound state, if it is not complexed to an exportin.
Therefore, Ran in GTP/GDP state triggers release of import/export cargo only in correct
location! It does not participate directly in binding, but rather only in release (and when
to release).
Summary: Importin Cycle
1. Ran-GTP displaces importin α from β and from cargo
2. Ran (with adaptor for α) exports importins back into cytosol
3. Ran’s GAP converts it to the GDP bound-state, which releases the importins
outside the nucleus Ran-GDP is returned to nucleus by its own import factor
Peroxisomes contain enzymes that use oxidation for metabolic purposes
Oxidation is used not for energy generation! (That only happens in the mitochondria).
They are single-membrane organelles, and generate H2O2 as an oxidizing agent, for fatty
acid degradation and synthesis of cholesterol-related lipids and some other lipids. They
are not part of the secretory pathway!
Peroxisomes can have a wide variety of forms—almost-empty “ghosts”, mature (and full)
peroxisomes through import of proteins, or fission into smaller peroxisomes.
Peroxisome Translocation
Membrane is impermeable (due to the toxic hydrogen peroxide inside)—small molecules
cannot diffuse in, unlike the nuclear membrane. Consequently, import is active and
requires specific protein complexes. However, if specific protein complexes were there,
large, oligomeric, or native proteins can be carted in directly. This is unlike translocation
into the ER (must be unfolded polypeptide), mitochondria, or nuclei (which is not sealed
at all).
Peroxisome Targeting signals
Most common are at the extreme C-terminus (SKL or similar) and recognized by major
receptor Pex5. Alternatively, there is one by the N-terminus (RLXXXXXHL), which is
recognized by the receptor Pex7. Transmembrane proteins are recognized by other
receptors. All peroxisome targeting receptors are soluble proteins that shuttle on and off
membrane from the cytosol.
Peroxisome Import is as follows:
1. Receptors are bound by docking complex on the membrane, and cross the
membrane together with the cargo
2. Release actors in the matrix/lumen trigger release of the cargo
3. The receptor left are bound by another complex in the membrane
a. An E3 ligase on the membrane monoubiquitinates the receptor
b. An AAA protein recognizes the ubiquitin and pulls the receptor out from
the peroxisome
4. Receptor is in the cytosol; DUB removes the ubiquitin and cycle restarts