Kontinkangas, L101A
Biochemistry of cellular organelles
Lectures: 1. Membrane channels;
2. Membrane transporters;
3. Soluble lipid/metabolite-transfer proteins;
4. Mitochondria as cellular organelles;
Seminar: Isolation of subcellular organelles;
5. Mitochondrial inheritance;
6. Mitochondria in health and disease;
7. Endoplasmic Reticulum (ER) and lipids;
8. Structure and function of peroxisomes;
Seminar: Mitochondria in cellular life.
Dr. Vasily Antonenkov, Visiting professor
Dept. Biochemistry, Oulu University
Oulu, Finland
Web site:
Lecture 7: Endoplasmic Reticulum (ER) and lipids
Lecture content:
Morphology of ER;
Overview of functions;
ER and protein synthesis;
ER and lipid synthesis;
Lipid secretion out of cells;
Dolichol and protein glycosylation;
ER stress.
Morphology of ER
• The most abundant membrane network in the cell, highly dinamic
• Rough (containing ribosomes) and smooth ER are membrane cisterna,
vesicles and tubules held together by cytosceleton;
• The cisternal space (or lumen, about 10% of the total cell volume) is
continuous with the periniclear space but separate from the cytosol;
• Smooth ER contacts physically with mitochondria and plasma
Protein sorting
• Proteins synthesized on free
ribosomes either remain in the
cytosol or are transported to
• Proteins destined to ER contain a signal
sequence at their amino (N) terminus;
• Most signal sequences contain a stretch
of hydrophobic aa, preceded by basic
The N terminus of growth hormone
• Proteins synthesized on
membrane-bound ribosomes are
translocated into the ER when
translation is in progress. They
may be either retained within the
ER or transported to peroxisomal
membrane or the Goldgi and, from
the the Goldgi apparatus, to
lysosomes, the plasma membrane,
or the cell exterior via secretory
Cotranslational targeting of proteins to ER
1. As the signal sequence emerges from the free ribosome, it is recognized and
bound by the signal recognition particle (SRP, heterooligomeric protein);
2. The SRP escorts the complex to the ER membrane where it binds to the SRP
3. The SRP is released, the ribosome binds to a membrane translocation complex
(Sec6) containing the channel protein that accomodates the signal sequence;
4. Translation resumes,and the growing chain
is translocated across the membrane;
5. Cleavage of the signal sequence by signal
peptidase releases the polypeptide into the
lumen of the ER.
Rough ER function
• The main function is the production and
processing of proteins that are later
exported from the cell or participate in
formation of plasma membrane, lysosomes
or peroxisomes;
• Two types of newly synthetized proteins:
(1) embedded into the membrane of ER,
(2) soluble in the lumen;
• Folding and modification of proteins
using chaperon proteins in the lumen,
initial glycosylation;
• Transport of newly synthetized proteins
mainly to the Golgi apparatus for further
processing and export out of the cell;
• Protein quality check - misfolded proteins
sent back for recycling.
Smooth ER functions
• Common:
• Lipid (including steroids) synthesis;
• Detoxificatrion – inactivation of harmful
toxins, like drugs and metabolic waste;
• Ca storage;
• Transport of the newly synthesized
proteins from rough ER by means of
membrane vesicles.
• Glucose-6-phosphatase –
• Reproductive organs – production of
steroid hormones.
Lipid synthesis in smooth ER
• Fatty acid elongation pathway
produces very long chain fatty
acids from palmitic acid
Synthesis of membrane lipids: phosphatidylcholine as an example
GPAT: Glycerol phosphate acyltransferase; LPAAT: Lysophosphatydic acid
CDP: cytidine diphosphate.
Lipid transfer :
-through contact sites;
-membrane receptors;
-membrane transporters
(ABC-proteins, others);
-vesicular transport;
Lipid traffic at membrane contact sites
• (a) The ER forms a network for transfer lipids between organelles by using
membrane contact sites (MCS), in these sites the lipid-transfer proteins (LTP) are
• (b) LTP is shuttling between membrane receptors in the MCS and transfer lipid
according to concentration gradient;
• (c) The LTP may be
simultaneously bound to
both receptors of the
opposite membranes of
MCS that would increase
the efficiency of lipid
• MCS are also important
to transfer Ca between
Transfer of membrane lipids from ER to other organelles and to
plasma membrane
Two main mechanisms:
The vesicles contain all
components characteristic for
membranes, including
phospholipids, cholesterol
and other lipids as well as
some membrane proteins. In
addition they can carry some
proteins and/or metabolites
confined inside the particles
(e.g., excretion of proteins
from epithelial cells during
milk production in mammary
Soluble lipid-transfer
proteins specific for
membrane lipids such as
phosphatidylcholine transfer
• Glycosylation is the reaction in which a carbohydrate (glycosyl donor) is
attached to a hydroxyl or other functional group of another molecule (lipids
or proteins);
•Protein glycosylation is a form of co-translational or post-translational
modification; it is important in function of plasma membrane, cell to cell
connection, activity of secreted proteins;
• N-glycosylation – carbohydrate attached to a nitrogen of asparagine or
arginine side-chains of proteins; it requires participation of a specific lipid –
dolichol phosphate; mainly occurs in the rough ER;
• O-glycosylation – carbohydrate attached to the hydroxy oxygen of serine,
threonine, tyrosine, hydroxyproline, or to oxygens on lipids such as
ceramide; mainly occurs in the Golgy apparatus;
• Phospho-glycosylation – carbohydrates linked through the phosphate to a
• Dolichyl-phosphate is
a glycosyl carrier lipid
for N-glycosylation;
• Dolichol is long
enough to span the
membrane 4-5 times; it
is localized on the
rough ER;
• The oligosaccharide
precursor is common to
most eukaryotes, but
after transfer to protein
can be modified.
Dolichyl-phosphate is formed by dolichol
CTP + dolichol > CDP + dolichyl phosphate
Formation of lipid-linked oligosaccharide (llo) precursor
The synthesis starts at the cytosolic face of the ER. (1-3) 2Acgl and 5 mannose
residues are added one at time to DolPP; (4) Than the product is flipped to the
luminal face; (5,6) In the latter reactions each mannose or glucose residue is
transferred from nucleotide sugar to dolichol on the cytosolic face of the ER, then it
is flipped and transferred to the growing oligosaccharide. The carrier (DolP) is then
flipped back again to the cytosolic face (catalyzed by flippases).
Transfer from dolichol to protein
• The oligosaccharide precursor is
transferred from the dolichol to an Asn
residue of the nascent protein, i.e.
• The Asn residue must be in a
sequence N-X-S/T;
• The process is catalyzed by
oligosaccharide protein transferase. The
enzyme is build up of three subunits.
Two of them are ER transmembrane
proteins. Their cytosolic parts bind to the
larger subunit of the ribosome (they act
as an anchor) The third subunit has the
catalytic activity.
Modifications of N-linked oligosaccharides
• After an oligosaccharide chain has been added to the protein, the 3 glucose
and 1 mannose residues are removed by three different enzymes in a fixed
• It occurs in the ER and is a signal that the protein is folded and can be
transported to the Golgi for further processing.
• The proper folding of the protein
is controlled by the enzyme:
glucosyl transferase (UGGT). If
protein is misfolded, the UGGT
reglucosylates it (adds glucose)
that prevents protein from
transfer to the Golgi.
Protein sorting in ER
• Addition of glucose to proteins by UGGT trigger interaction of them with
lectins – group of proteins able to interact with protein-bound sugars, such
as calreticulin (CRT) and calnexin (CNX);
• Interaction with CRT or CNX
prevents from transfer of misfolded
proteins from ER to the Golgi;
• Delay in the transfer to the Golgi of
misfolded proteins increases the
probability of their recognition by
glucose regulated protein 78
(Grp78) or EDEM protein that lead
to excretion of misfolded proteins
from ER.
Endoplasmic reticulum-associated degradation (ERAD)
• The misfolded proteins are prone to aggregate and hence course an ER
• Some membrane-bound chaperones (i.e. EDEM protein) guide the
retrotranslocation of the misfolded protein back into the cytosol;
• In the cytosol the misfolded
protein is ubiqutinated and
subjected to proteasomal
• The nature of the membrane
machinery catalyzing export of
misfolded proteins is not clear.
ER stress
The consequence of a mismatch between the
load of unfolded and misfolded proteins in the
ER and the capacity of cellular machinery to
cope with that.
• The ER stress may results from deficiency of cell metabolism,
i.e. deficit in energy supply, oxidative stress, etc.;
• The ER stress leads to Unfolded Protein Responce (UPR).
Unfolded protein response (UPR)
• UPR is an adaptive regulatory
response of the cell to ER stress;
• 3 mechanisms in mammals: ATF6,
PERK, and IRE1;
• All mechanisms eventually affect
transcriptional (translational)
regulation of proteins synthesis;
• Increase in ER protein folding
• Decrease in ER-dependent protein
synthesis and folding.
• IRE1 is a threonine/serine kinase and promotes cytoplasmic splicing of
XBP1 mRNA, XBP1 is a transcriptional factor;
• PERK is a protein kinase and active in attenuation of protein synthesis;
• ATF6 is a transcription factor which undergoes maturation in ER;
• All receptors somehow sense misfolded proteins in the ER lumen and
transduce the signal to cytosol.
ATF6-dependent pathway
• ATF6 is a transcriptional factor that is initially
synthesized as an ER-resident transmembrane
• Upon accumulation of misfolded proteins, it is
packaged into transport vesicles that deliver it from
ER to the Golgi;
• In the Golgi the ATF-6 protein is cleaved by two
specific proteases leading to liberation of the Nterminal cytosolic domain ATF6(N);
• ATF6(N) moves into nucleus to activate UPR target
PERK pathway
• PERK is an ER-resident transmembrane
• When activated upon unfolded proteins,
PERK oligomerizes and phosphorylate itself
and translation initiation factor elF2 that
inactivates it;
• Hence, translation of most proteins
decreases that helps cope with the influx of
proteins in ER;
• However, some proteins, including
transcription factor ATF4, are preferably
translated when elF6 is limiting;
• ATF4 induces transcription of UPR target
IRE1 pathway
• IRE1 is a bi-functional enzyme
(kinase/RNase) in the membrane of ER;
• Due to binding the unfolded proteins,
the IRE1 is oligomerized that activates
kinase and endoribonuclease (RNase)
• RNase is active towards mRNA coding
for transcription factor XBP1. It cleaves
out one intron of this mRNA and after
ligation (by other enzymes) this RNA
translate XBP1;
• XBP1 moves into nucleus to activate
UPR target genes.
Suggested questions
• Morphology of ER;
• Sorting of the newly synthesized proteins and targeting of then into ER;
• Functions of rough and smooth ER;
• What kind of lipids are synthesized in ER and how (example);
• What mechanisms of the lipid traffic do you know? Transfer of lipids at
membrane contact sites;
• Transfer of lipid from ER to other organelles and to plasma membrane;
• N- and O-glycosylation of proteins in ER, role of dolichol;
• Step-by-step mechanism of N-glycosylation;
• Sorting folded from unfolded proteins in ER;
• ER stress – what is it? Unfolded protein response – its functional role;
• Three main mechanisms of the unfolded protein response –describe shortly
Thank you!

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