Year I/Committee I; Structure and Organization of the Cell
MEDICAL BIOLOGY and GENETICS
Protein Sorting and Transport
Asst. Prof. Dr. Oya Akça
Istanbul Atlas University-Faculty of Medicine
Department of Medical Biology and Genetics
2023-2024
Non-secreted proteins
Free ribosomes→nucleus,mito.,per., chl.
Secretory pathway
Rough ER →Golgi →Secretory vesicles→Out of the cell
• Proteins synthesized in free ribosomes remains in the cytosol or transported to nucleus, mitochondria, chloroplasts and
peroxisomes
• However, proteins synthesized in ribosomes bound to the ER membrane passes into the ER while translation is still in
progress, and also has a signal sequence for translocation
Targeting Proteins to the Endoplasmic Reticulum
Proteins can be transported into
the ER while their synthesis
continues on ribosomes attached
to the ER (cotranslational
translocation) ,
or they can be taken up into the
ER after synthesis is completed
on free ribosomes in the cytosol
(posttranslational translocation).
• In mammalian cells, the
import of proteins into
the ER begins before
the polypeptide chain is
completely
synthesized—
that is, import is a cotranslational process.
• This
distinguishes
the
process from the import of
proteins into mitochondria,
chloroplasts, nuclei, and
peroxisomes,
which
are posttranslational process
es.
Targeting Proteins to the Endoplasmic Reticulum, (membrane bound)
• The first step of the transport pathway during translation is attachment of the
ribosome-mRNA complex to the ER.
• Ribosomes are targeted to the ER by sequences in the synthesized polypeptide chain
• Free and membrane-bound ribosomes are functionally indistinguishable, and
all protein synthesis initiates on ribosomes that are free in the cytosol.
• Ribosomes engaged in the synthesis of proteins that are destined for
secretion are then targeted to the endoplasmic reticulum by a signal
sequence at the amino terminus of the growing polypeptide chain.
• These signal sequences are short stretches of hydrophobic amino acids that are
cleaved from the polypeptide chain during its transfer into the ER lumen.
Targeting Proteins to the Endoplasmic Reticulum (membrane bound)
• In the mechanism that targets the proteins to be
secreted to the ER during their synthesis, the
signal sequence is approximately 15-40 aa long
and contains a region of 7-12 hydrophobic aa.
Targeting Proteins to the Endoplasmic Reticulum, (membrane bound)
Step 1: As soon as the signal sequence is
synthesized in the ribosome, it is
recognized and bound by the signal
recognizing particle (SRP).
Step 2: SRP directs the polypeptide chain,
where it binds to the SRP receptor.
Step 3: The SRP is released, the ribosome
binds to the translocon, and the insertion
of the signal sequence into the channel
allows the translacon to open.
Step 4: translation continues and the
signal sequence is removed by the signal
peptidase.
Step 5: During the ongoing translation, the
elongated
polypeptide
chain
is
transported to the other side of the
membrane, into the ER lumen.
Step 6: The synthesized polypeptide chain
is released in the ER lumen.
Targeting Proteins to the Endoplasmic Reticulum, (free ribosomes)
Proteins which will be transported into
the ER after translation are
synthesized by free ribosomes and
held in unfolded conformation by
cytosolic chaperones.
SRP is not required for posttranslational transfer of targeted
proteins to the ER.
Signal sequences are recognized by
the Sec62/63 complex, the receptor
protein associated with translocons,
rather than SRP.
Targeting Proteins to the Endoplasmic Reticulum,
(free ribosomes)
The Hsp70 and Hsp40 chaperones in
the cytosol are required to keep the
polypeptide chain in an unfolded
form. Thus, these proteins can enter
the translocon.
The other Hsp70 (BiP) found in the
ER is required to pull the
polypeptide chain from the channel
into the ER.
The binding of multiple Bip proteins
and their release by ATP hydrolysis
provide the energy required for the
translocation of the polypeptide
chain into the ER.
• Insertion of Proteins into the ER Membrane
Proteins destined for secretion or
residence within the lumen of
the ER, Golgi apparatus, or
lysosomes are translocated across
the ER membrane and released into
the lumen of the ER
However, proteins destined for
incorporation into the plasma
membrane or the membranes of
the ER, Golgi, or lysosomes are
initially inserted into the ER
membrane instead of being
released into the lumen.
Insertion of Proteins into the ER Membrane
• From the ER membrane, they proceed
to their final destination along the
same pathway as that of secretory
proteins:
• ER ~ Golgi ~plasma membrane or ~
lysosomes.
• These proteins are transported along
this
pathway
as
membrane
components rather than as soluble
proteins.
• SOLUBLE PROTEINS
The peptide moves through the translocation
channel into the lumen of the ER. The signal
peptide sequence remains attached to the
membrane. It is later cleaved off by a signal
peptidase. Leaving the protein free in the lumen of
the ER.
• MEMBRANE PROTEINS
Key point is that the orientation of a
protein in the membrane is
established when it is first inserted
into the ER membrane.
This orientation of the protein persists
all of the way to its final destination.
That is, the cytosolic side of
membrane remains on the cytosolic
side throughout all processes.
As membrane proteins are being
translated, they are translocated or
transferred into the ER until a
hydrophobic membrane crossing
domain is encountered.
This serves as a 'stop transfer' signal
and leaves the protein inserted in
the ER membrane.
Protein Folding and Processing in the ER
• Protein folding and processing
occur either during translocation
across ER or within ER lumen.
• Lumenal ER proteins assist
folding
and
assembly
of
translocated polypeptides
• Hsp70 chaperone BiP binds to
unfolded polypeptide chain as it
crosses membrane, helps fold
and assemble complexes
• Disulfide (S—S) bond formation
facilitated by PDI (protein
disulfide isomerase)
Protein Folding and Processing in the ER
• Glycosylation: occurs on
specific asparagine (Asn)
residues
as
protein
translocates into ER.
• Oligosaccharide
is
synthesized
on
lipid
(dolichol) carrier.
• Glycosylation
prevents
protein aggregation in ER,
provides signals for sorting
Protein Folding and Processing in the ER
• Some proteins are anchored
to the plasma membrane not
by transmembrane domains
but via glycolipids.
• Glycosylphosphatidylinositol
(GPI) anchors
• GPI anchors assemble in ER
membrane, add to Cterminal Asn
• GPI-anchored proteins are
transported as membrane
components via secretory
path.
Quality Control in the ER
• Many of the proteins synthesized
in the ER are rapidly degraded.
This is mainly due to the inability
of proteins to fold correctly.
• Chaperones and sensors in ER
identify misfolded proteins, divert
them to degradation pathway.
• Ex. chaperone calreticulin assists
glycoproteins folding
• Protein folding sensor passes
correctly folded glycoproteins on
to transitional ER
Unfolded protein response (UPR)
• The level of unfolded protein is constantly monitored. This process is regulated
by the unfolded protein response (UPR) signaling pathway, which is activated by
the accumulation of unfolded protein in the ER.
• BiP plays role as sensor for protein folding.
• If excess of unfolded proteins accumulates, BiP initiates UPR:
• Includes general inhibition of protein synthesis, increased expression of
chaperones, increase in degradation of mRNAs
• If these changes fail to normalize the level of unfolded protein in the ER,
activation of the unfolded protein response pathway triggers programmed cell
death. Thus, cells that cannot fold the synthesized proteins correctly are
eliminated.
• Vesicles export proteins and
phospholipid molecules from
ER, bud from transitional ER,
move
through
ER-Golgi
intermediate
compartment,
and then to Golgi.
• Proteins in lumen of one
organelle bud into transport
vesicles, release to lumen of
recipient organelle by vesicle
fusion.
• Membrane proteins and lipids
are transported in similar way;
topological
orientation
is
maintained.
• The Endoplasmic Reticulum Return to the ER: Some proteins must stay in ER (BiP,
signal peptidase, etc). Target sequence (KDEL or KKXX) at carboxy terminus
directs retrieval from ERGIC or Golgi complex via recycling path.