Protein targeting
or
Protein sorting
Refer Page 1068 to 1074
Principles of Biochemistry by Lehninger
& Page 663 Baltimore Mol Cell Biology
• Protein targeting or Protein sorting is the
process of delivery of newly synthesized
proteins to their proper cellular destinations
• Proteins are sorted to the endoplasmic reticulum
(ER), mitochondria, chloroplasts, lysosomes,
peroxisomes, and the nucleus by different
mechanisms
• The process can occur either during protein
synthesis or soon after synthesis of proteins by
translation at the ribosome.
• Most of the integral membrane proteins,
secretory proteins and lysosomal proteins
are sorted to ER lumen from where these
proteins are modified for further sorting
• For membrane proteins, targeting leads to insertion of
the protein into the lipid bilayer
• For secretory/water-soluble proteins, targeting leads to
translocation of the entire protein across the membrane
into the aqueous interior of the organelle.
• Protein destined for cytosol simply remain where they are
synthesized
• Mitochondrial and chloroplast proteins are first
completely synthesized and released from ribosomes.
These are then bound by cytosolic chaperone proteins
and delivered to receptor on target organelle.
• Nuclear proteins such as DNA and RNA polymerases,
histones, topoisomerases and proteins that regulate gene
expression contain Nuclear localization signal (NLS)
which is not removed after the protein is translocated.
Unlike ER localization signal sequence which is at N
terminal, NLS ca be located almost anywhere along the
primary sequence. Most NLS consist of four to eight amino
acid residues with consecutive basic (Arg or Lys) residues
• Proteins sorted to ER contains amino terminal
Signal sequence which translocates these
proteins to lumen of ER
• The function of Signal Sequence was first
proposed by G Blobel in 1970
• The signal sequence can be 13 to 36 amino acids
residues
– 10 to 15 residues are hydrophobic amino acids
– There is one or more positively charged amino acid
near amino terminal preceding hydrophobic residues
– A short polar sequence at the carboxyl terminus near
cleavage site (eg Ala residue)
• George Palade demonstrated that proteins with
ER signal sequence are synthesized on
ribosomes attached to ER (rough ER) and Signal
sequence helps to direct ribosomes to ER
Directing eukaryotic proteins with the appropriate signals to the
Endoplasmic Reticulum
Directing eukaryotic proteins with the appropriate signals to the Endoplasmic
Reticulum
• The protein targeting pathway begins with initiation of
protein synthesis on free ribosomes. The ER signal
sequence appears early in protein synthesis because it is
at the amino terminus.
• Once the ER signal sequence emerges from the ribosome,
it is bound by a signal-recognition particle (SRP)
• The SRP delivers the ribosome/nascent polypeptide
complex to the SRP receptor in the ER membrane. This
interaction is strengthened by binding of GTP to both the
SRP and its receptor
• Transfer of the ribosome/nascent polypeptide to the
translocon (peptide translocation complex) leads to
opening of this translocation channel and insertion of the
signal sequence and adjacent segment of the growing
polypeptide into the central pore
• Both the SRP and SRP receptor, once
dissociated from the translocon, hydrolyze their
bound GTP and then are ready to initiate the
insertion of another polypeptide chain
• As the polypeptide chain elongates, it passes
through the translocon channel into the ER
lumen, where the signal sequence is cleaved
by signal peptidase and is rapidly degraded
• Once translation is complete, the ribosome is
released, the remainder of the protein is drawn
into the ER lumen, the translocon closes, and
the protein assumes its native folded
conformation
Glycosylation Plays a Key Role in Protein Targeting
• Following the removal of signal sequences, polypeptides
are folded, disulfide bonds formed, and many proteins
glycosylated to form glycoproteins
• In many glycoproteins the linkage to their
oligosaccharides is through Asn residues.
• These N-linked oligosaccharides are diverse, but the
pathways by which they form have a common first step.
• A 14 residue core oligosaccharide is built up in a
stepwise fashion, then transferred from a dolichol
phosphate donor molecule to certain Asn residues in
the protein
• The transferase is on the lumenal face of the ER and
thus cannot catalyze glycosylation of cytosolic proteins
• After transfer, the core oligosaccharide is trimmed. All Nlinked oligosaccharides retain a pentasaccharide core
derived from the original 14 residue oligosaccharide.
Dolichol phosphate
• A few proteins are O-glycosylated in the ER, but most Oglycosylation occurs in the Golgi complex or in the cytosol
• Several antibiotics act by interfering with one or more steps
in this process and have aided in elucidating the steps of
protein glycosylation. The best-characterized is
tunicamycin, which mimics the structure of UDP-Nacetylglucosamine and blocks the first step of the process
• The presence of one or more mannose 6-phosphate
residues in its N-linked oligosaccharide is the structural
signal that targets the protein to lysosomes
• A receptor protein in the membrane of the Golgi complex
recognizes the mannose 6-phosphate signal and binds
the hydrolase so marked.
• Vesicles containing these receptor-hydrolase complexes
bud from the trans side of the Golgi complex and make
their way to sorting vesicles.
• Here, the receptor-hydrolase complex dissociates in a
process facilitated by the lower pH in the vesicle and by
phosphatase-catalyzed removal of phosphate groups from
the mannose 6-phosphate residues.
• The receptor is then recycled to the Golgi complex, and
vesicles containing the hydrolases bud from the sorting
vesicles and move to the lysosomes.
Phosphorylation of mannose
residues on lysosome-targeted
enzymes. N-Acetylglucosamine
phosphotransferase recognizes
some as yet unidentified
structural feature of hydrolases
destined for lysosomes.
Bacteria Also Use Signal Sequences
for Protein Targeting
• Bacteria can target proteins to their inner
or outer membranes, to the periplasmic
space between these membranes, or to
the extracellular medium. They use signal
sequences at the amino terminus of the
proteins
• Most proteins exported from E. coli make
use of the following pathway
Bacteria Also Use Signal Sequences
for Protein Targeting
• Model for protein export in bacteria.
• 1 A newly translated polypeptide binds to the cytosolic chaperone
protein SecB, which
• 2 delivers it to SecA, a protein associated with the translocation
complex (SecYEG) in the bacterial cell membrane.
• 3 SecB is released, and SecA inserts itself into the membrane,
forcing about 20 amino acid residues of the protein to be exported
through the translocation complex.
• 4 Hydrolysis of an ATP by SecA provides the energy for a
conformational change that causes SecA to withdraw from the
membrane, releasing the polypeptide.
• 5 SecA binds another ATP, and the next stretch of 20 amino acid
residues is pushed across the membrane through the translocation
complex.
• Steps 4 and 5 are repeated until 6 the entire protein has passed
through and is released to the periplasm.
• The electrochemical potential across the membrane (denoted
by + and - ) also provides some of the driving force required for protein
translocation.