Download Intracellular Compartments

Intracellular Compartments
The Endomembrane System
The Endomembrane System
• A “continuous” System of membrane bound
organelles:
• Nuclear Envelope
• Endoplasmic Reticulum
• Golgi Complex
• Endosomes
• Lysosomes
The Endomembrane System
• Endoplasmic Reticulum and Golgi Complex: Protein
synthesis, processing and sorting
• Early endosomes: sorting of materials brought into
the cell by endocytosis
• Late endosomes: Mature into lysosomes
• Lysosomes: contain enzymes capable of breaking
down macromolecules
• Materials move between the endomembrane
compartments through/by transport vesicles
The Endoplasmic Reticulum
• Endoplasmic Reticulum (ER) a continuous network
of flattened sacs, tubules, and vesicles.
• The sacs are called cisternae, the space enclosed is
the ER Lumen.
• The ER is continuous with the nuclear membrane, so
material in the ER lumen can move freely into the
perinuclear space (between the two layers of the
nuclear envelope)
• Up to 90% of a cell’s membrane may be in the ER
The E R : Functions
• Contains enzymes necessary for biosynthesis
of proteins of the ER, Golgi, endosomes,
lysosomes, plasma membrane
• Biosynthesis of proteins secreted by the cell
• Biosynthesis of lipids: triacylglycerols and
cholesterol
• The source of most of the lipids that are
assembled into intracellular and plasma
membranes
The E R
• Smooth ER and rough ER: rough ER has
attached (on the cytosolic surface) ribosomes
• Rough ER is flattened sacs, smooth ER is
tubules
• Transitional elements: portion of the rough ER
which forms transition vesicles (shuttle material
to the Golgi): looks like smooth ER
• Smooth and Rough ER are continuous one with
the other: material can travel from one to the
other
The E R
• Cells involved in protein secretion (like the
liver) have extensive rough ER networks
• Cells involved in steroid production have
extensive smooth ER networks
• Homogenization of cells to purify subcellular
components breaks the ER into small vesicles
called microsomes: these have been important
to studying ER function, but do not exist in the
cell
The Rough ER
• The ribosomes on the surface of the ER are
responsible for synthesis of both membrane-bound
(organelle and plasma membrane proteins) and soluble
proteins (organelle and secreted proteins)
• Most proteins that enter the endomembrane system
enter the ER cotranlationally, that is, as translation is
occurring
• Most proteins of other membrane bound organelles
(mitochondria, chloroplasts, peroxisomes) are
transported there posttranslationally.
The Rough ER
• The rough ER is the site for:
• Initial steps of carbohydrate addition
(glycosylation)
• Folding of proteins
• Assembly of multimeric proteins
• “Quality control”: improperly folded or
modified proteins are retained or degraded
The Smooth ER
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The smooth ER is involved in:
Drug detoxification
Carbohydrate metabolism
Calcium storage
Steroid biosynthesis
Membrane biosynthesis
Smooth ER: Hydroxylation Reactions
• Hydroxylation reactions (addition of –OH group to an
organic molecule) are important in drug detoxification
(the hydroxylated form is more water soluble, and can
be eliminated from the body) and in steroid
biosynthesis.
• These reactions depend on a cytochrome P-450 and
NADPH
• 1) an electron transport system (in the smooth ER)
transfers e- from NADPH to cytochrome P-450
• 2) Reduced cytochrome P-450 donates an electron to
O2, activating it
• 3) O2 is reduced to H2O, -OH is added to the organic
molecule
Smooth ER: Hydroxylation Reactions
• Hydroxylation of fatty acids uses NADH
(instead of NADPH) as an electron source
• Enzymes which carry out these reactions (one
atom of the O2 is added as a hydroxyl, the other
is reduced to H2O) are called mixed-function
oxidases, or monooxygenases
• Mixed function oxidases can hydroxylate
numerous compounds, and are upregulated by
toxic compounds
Smooth ER: Hydroxylation Reactions
• The behavior of oxidases explains several
physiological observations:
• Up-regulation (increased smooth ER) due to exposure
to a toxic drug (example, phenobarbitol) results in
habituation, larger and larger doses of the drug are
required to achieve the same effect since the body is
able to more efficiently degrade the drug.
• The ability to act on multiple substrates explains the
observation that exposure to one drug can reduce the
effectiveness of other unrelated drugs (example,
antibiotics in barbituate users)
Smooth ER: Hydroxylation Reactions
• A different hdroxylation reaction, involving cytochrome
P-448 and the enzyme aryl hydrocarbon hydroxylase, is
involved in metabolizing polycyclic hydrocarbons: those
containing two or more benzene rings (these are
particularly toxic/ carcenogenic)
• The oxidised products of this reaction are often more
toxic than the original compound; aryl hydrocarbon
hydroxylase can convert potential carcinogens into their
active forms
• Mice producing high levels of aryl hydrocarbon
hydroxylase have high levels of spontaneous tumor
formation
• Cigarette smoke induces aryl hydrocarbon hydroxylase
Smooth ER:
• Carbohydrate metabolism: The transfer of glucose from
glycogen (in liver cells) in response to hormones (via
the cAMP signal transduction pathway) is closely linked
to smooth ER enzymes, such as the glucose-6phosphatase, which removes the phosphate from
glucose-6-phosphate (from glycogen breakdown)
making it available for export (by a glucose transporter)
• Calcium storage: smooth ER in some cells stores high
levels of Ca+2, by using an ATP dependent Ca+2 pump:
Ca+2 can be rapidly released when needed (such as
activation of IP3 signaling pathway)
Smooth ER:
• Biosynthesis of membranes: Most of the membrane
phospholipids are made exclusively in the smooth ER
• The enzymes involved in phospholipid synthesis are
located on the cytosolic face of the smooth ER:
phospholipids are added to the cytosolic leaf, then
translocated (flipped) to the inner leaf by an enzyme.
• Components of the endomembrane system get lipid by
fusing to vesicles from the ER.
• Mitochondria and chloroplasts (and the other
membranes) get lipids by the action of phospholipid
exchange proteins, which carry specific phospholipids
from the ER, through the cytosol, to the organelle
membrane.
Golgi Complex
• The Golgi Complex is a series of compartments
involved in protein modification, sorting, and
secretion. The major divisions of the Golgi are:
• Cis-Golgi network: closest to ER
• Medial Golgi network: in the middle
• Trans-Golgi network: farthest from ER, closest
to cell surface
• Transport vesicles
Movement Through the Golgi
• Vesicles “bud” from the ER, fuse with the
Golgi
• Vesicles move from ‘layer’ to layer of the
Golgi
• Vesicles are moving in both directions, from
ER toward cell surface and from trans-golgi
toward ER
Golgi Processes
• Modification of N-linked oligosaccharides
• O-linked glycosylation
• Synthesis of oligosaccharides (such as
extracellular matrix components)
• Protein sorting: proteins are selectively
transported ‘outward’ (anterograde) or ‘inward’
(retrograde). Signals in the protein and
receptors in the membrane are involved in this
process.
N-linked Glycosylation
• This is the attachment of oligosaccharides to the
amino group of certain asparagine residues in a
protein.
• There are two major steps: attachment of the
“core oligosaccharide” and modification of the
oligosaccharide.
• Core glycosylation occurs in the ER.
• Most of the modifications occur in various levels
of the Golgi.
N-linked Glycosylation
• Attachment of the core oligosaccharide occurs in the
ER: the core is built on the ER membrane,. Attached to
a lipid called dolichol phosphate.
• After partial synthesis on the outside of the ER
membrane, the glycosylated dolichol phosphate is
flipped so the oligosaccharide faces the inside of the
ER.
• The Oligosaccharide is further extended, and then
transerred to the –N of the glycosylation site of the
protein.
• Specific glucose and mannose residues are then
removed from the core oligo in the ER.
N-linked Glycosylation
• As the protein is carried through the Golgi,
additional sugars may be added to the core
oligosaccharide.
• The modification pattern varies from one
protein to the next, some proteins are not
modified, others have extensive additions of Nacetylglucosamine, galactose, sialic acid, and
fucose.
O-linked Glycosylation
• Carbohydrate groups are also added to the –O
of serine, threonine, and hydroxyproline
residues as the protein passes through the
Golgi.
• One of the ways the movement of proteins
through the golgi has been studied is by
identifying the modifications occurring in
various compartments.
• Proteins can be recovered in the ER with
modifications which are only carried out in the
Golgi, demonstrating the retrograde motion.
Protein Sorting
• Proteins contain specific sequences that signal
to the cell’s machinery what the fate of the
protein is to be.
• For example, the sequence KDEL (lysine,
aspartate, glutamate, leucine) at the carboxy
terminus of a protein signals that it should be
retained in the ER (not transported out).
• However, some ER proteins are O
glycosylated, indicating that they have been
in the Golgi.
Protein Sorting
• Retention of KDEL proteins appears to involve
receptors for the KDEL sequence in membrane
proteins of the Golgi.
• Binding of the KDEL changes the conformation
of the receptor, somehow triggering the membrane
to incorporate into vesicles which are transported
back to the ER.
• Once fused with the ER, the pH is slightly more
acidic than in the Golgi, which triggers a change
in the Km of the KDEL sequence for the receptor,
and the KDEL protein is released into the ER
lumen.
Protein Sorting
• Another method of protein sorting appears to
depend on the length of membrane spanning
regions of integral membrane proteins.
• The thickness of the membrane increases as you
move from the ER to the plasma membrane (from
5 nm to 8 nm)
• There is a correlation between the length of the
hydrophobic stretches of the membrane proteins
and the thickness of the membrane of the Golgi
compartment in which the protein ends up.
Protein Targeting
• Proteins that move out of the Golgi must be
targeted to end up in the right place (endosomes,
lysosomes, secretory vesicles etc.)
• Soluble lysosomal proteins are targeted by
recognition of mannose-6-phosphate in the
carbohydrate side chains by specific receptors on
the membrane of trans-Golgi compartment (pH in
trans-Golgi is about 6.4).
• These receptor/protein complexes are then
packaged into clathrin coated transport vesicles,
and conveyed to an endosome.
Protein Targeting
• The endosome is called an early endosome.
• As the endosome ‘matures’, the pH inside it
decreases to about 5.5
• At this low pH, the lysosomal proteins (mannose6-phosphate containing) release from the receptor
and become soluble in the endosome.
• The receptors are recycled via vesicles that fuse to
the Golgi.
• The late endosome matures into a lysosome or
fuses with a lysosome to deliver the contents
(digestive proteins)