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

• 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

Directing eukaryotic proteins with the appropriate signals to the Endoplasmic Reticulum

Directing eukaryotic proteins with the appropriate signals to the Endoplasmic Reticulum

• 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

• 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 N-linked oligosaccharides retain a pentasaccharide core derived from the original 14 residue oligosaccharide.

•

Glycosylation Plays a Key Role in Protein Targeting

Dolichol phosphate

• A few proteins are O-glycosylated in the ER, but most O-glycosylation 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-N-acetylglucosamine 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-Acetylglucosaminephosphotransferase recognizes some as yet unidentifiedstructural feature of hydrolases destined for lysosomes.

Bacteria Also Use Signal Sequencesfor 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 Sequencesfor 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.