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Protein Sorting in the Cell
Eukaryotic cells cytoplasm contain many different specialized compartments called………………….. organelles
Each organelle contains a distinct set of proteins that mediates its unique functions.
Proteins within eukaryotes are sorted by two basic types of protein targeting pathways
PROTEIN TARGETING AND SORTING
• Post‐translational pathway targets newly synthesized proteins to their appropriate compartment. This pathway is utilized by proteins destined to the nucleus, mitochondria, chloroplast and peroxisome.
• Co‐translational pathway involves the co‐translational transport of a protein into the endoplasmic reticulum lumen. This pathway is utilized by proteins destined for the ER, Golgiapparatus, lysosome, or the plasma membrane. Secreted proteins are also produced and targeted by this mechanism.
After translation, proteins move to various compartment by:
(i)gated transport(ii)trans‐membrane transport, (iii)vesicular transport.
Key elements for the Protein targeting events
Signal sequencesShort regions of a protein that act as targeting signals to direct the protein to specific subcellular localization
Receptors that recognize particular signal sequences
Translocation channels that allow transfer ofproteins across the membrane bilayer.
Require energy (ATP or GTP)
Examples of different types of signal sequence
These sequences function like cellular Postal Codes targeting proteins towardstheir destination in the cell.
(A)Signal sequences may be present in N‐terminal or
(B) distributed within the protein sequence which fold to form a SIGNAL PATCH.
The Transport of Molecules into and out of the Nucleus
Many proteins that function in the nucleus including histones, DNA and RNA polymerases, gene regulatory proteins, and RNA‐processing proteins –are selectively imported into the nuclear compartment from the cytosol.
At the same time, tRNAs and mRNAs are synthesized in the nuclear compartment and then exported to the cytosol.
Nuclear transport involves the passage through a “gate” that separates two aqueous compartments,the cytoplasm and the nucleoplasm.
Small molecules (5000 daltons or less) can diffuse freely through the nuclear pore, larger molecules require active transport.
Larger proteins bind to specific receptor proteins located in the pore complexes and are then actively transported across the nuclear envelope through the complexes.
The nuclear envelope of a typical mammalian cell contains 3000 to 4000 nuclear pore complexes.
If the cell is synthesizing DNA, it needs to import about 106 histone molecules from the cytosol every 3 min in order to package newly made DNA into chromatin.
Key element for nuclear transport
1. Nuclear Localization Sequence (NLS)
2. Nuclear pores complex
3. Transport protein (i) Importins: Cytoplasm to nucleus(ii) Exportins: Nucleus to cytoplasm
For proteins too large to diffuse through the Nuclear Pore, an Nuclear Localization Sequence(4‐8 aa length) is required for nuclear import
Nuclear Transport: Cytoplasm to Nucleus
Step 1. Protein containing NLS binds soluble NLS receptor importin at the cytoplasmic side
Step 2 Importin:NLS protein complex transports to cytoplasmicfilaments
Step 3. Cytoplasmic filaments bend toward nucleus
Step 4. Change in conformation of transporter
Step 5. Inside the nucleus, Importin:NLS protein complex interaction with Ran-GTP causes a conformational change in the importin that causes dissociation
Nuclear export roughly reverses the import process; in the nucleus, the Exportinbinds the cargo and Ran‐GTP and diffuses through the pore to the cytoplasm, where the complex dissociates.
Nuclear Transport: Nucleus to Cytoplasm
The GTP binding protein Ran regulates Nuclear Transport
EXPORTIMPORT
Importins Exportins
Protein with NLS
Protein with NES
The Ran‐GTP/GDP cycle
Ran‐GAP‐Ran‐GTPase‐accelerating proteinRan GEF‐Ran‐guanine nucleotide–exchange factor
The Transport of Proteins into Mitochondria and Chloroplasts
Mitochondria and chloroplasts are double membrane enclosed organelles.
Both organelles contain their own DNA, ribosomes, and other components required for protein synthesis, most of their proteins are encoded in the cell nucleus and imported from the cytosol.
There are two subcompartments in mitochondria: the internal matrix space and the intermembrane space.
Chloroplasts have the same two subcompartments plus an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins.
Most of the mitochondrial precursor proteins have a signal sequence at their N‐terminus that is rapidly removed after import by a protease (the signal peptidase) in the mitochondrial matrix.
When the signal sequence is folded as an α helix, the positively charged residues (red) are seen to be clustered on one face of the helix, while the nonpolar residues (yellow) are clustered primarily on the opposite face.
Protein translocation from the cytoplasm to the mitochondrial matrix requires two distinct translocation complexes Tom & Tim: the TOM complex functions across the outer membrane, and two TIM complexes, the TIM23 and TIM22 complexes, function across the inner membrane.
Cytoplasmic proteins cross both the outer and inner mitochondrial membranes in a single step:
TOM complex: It recognizes the signal sequences of all nucleus‐encoded mitochondrial proteins and import into the intermembrane space.
TIM23 complex: It transports proteins into the matrix space.
TIM22 complex: It mediates the insertion of a subclass of inner membrane proteins, including the carrier protein that transports ADP, ATP, and phosphate.
OXA complex: It mediates the insertion of inner membrane proteins that are synthesized within the mitochondria in the inner mitochondrial membrane. It also helps to insert some proteins that are initially transported into the matrix by the TOM and TIM complexes.
ATP hydrolysis and a H+ gradient are used to drive protein import into mitochondria.
Protein import into the mitochondrial matrix is a stepwise process:
1)Bound cytosolic hsp70 is released from the protein in a step that depends on ATP hydrolysis. After initial insertion of the signal sequence and of adjacent portions of the polypeptide chain into the TOM complex, the signal sequence interacts with a TIM complex.2)The signal sequence is then translocated into the matrix in a process that requires an electrochemical H+ gradient across the inner membrane, positioning the unfolded polypeptide chain so that it transiently spans both membranes. 3)Mitochondrial hsp70 binds to regions of the polypeptide chain as they become exposed in the matrix, thereby “pulling” the protein into the matrix. ATP hydrolysis then removes the mitochondrial hsp70, allowing the imported protein to fold.
Protein transport into chloroplasts
It resembles transport into mitochondria in many respects. Both processes occur posttranslationally, use separate translocation complexes in each membrane, occur at contact sites, require energy, and use N‐terminal signal sequences. Proteins are transported from the cytosol to their final destination in two steps.
1.First, they pass across the double membrane at contact sites into the matrix space of the chloroplast, called the stroma, and
2.Secondly, they are translocated into the thylakoidmembrane (or across this membrane into the thylakoid space)
Translocation into the thylakoid space or thylakoidmembrane can occur by any one of at least four routes:
1. Sec pathway,2. SRP (signal recognition particle)‐like pathway,3. ΔpH pathway,4. Spontaneous insertion pathway
The Transport of Proteins into Peroxisome
Peroxisomes
Cytoplasmic organelles
Bounded by a single membrane.
Used by the cell as a place to sequester specific reactions.
These often generate hydrogen peroxide
Import of peroxisomal matrix proteins is post‐translational
One well defined peroxisomal import signal is the sequence Ser‐Lys‐Leu at the extreme C‐terminus of a protein.
Mechanism of import is not well‐defined.
Transport into the Endoplasmic Reticulum(Gateway to the Secretory Pathway)
The “Rough” ‐Endoplasmic Reticulum with ribosomes attached is the site of co‐translational translocation of proteins into the ER
In co‐translational translocation, the nascent protein crosses the ER membrane as it leaves the ribosome
ER targeting signals are recognized by SRP(Signal Recognition Particle)
SRP recognizes signal sequences as they come off the ribosome and carries the mRNA‐ribosome‐nascent polypeptide complex to the ER
SRP complex then binds to SRP receptor . Binding to receptor results translocation of polypeptide to ER lumen whereas translation continues . SRP and SRP receptor later dissociates and start fresh cycle.
Translocation of a soluble protein into the ER lumen
Integral membrane proteins contain “stop transfer”sequences that lead to release of the protein into the ER membrane
Once proteins have entered the endoplasmic reticulum they can be move on to other compartments of the secretory pathway or out of the cell entirely.
But in addition to acting as a port of entry for proteins into the secretory pathway, two additional important functions for protein targeting take place in the ER.
‐N‐linked glycosylation of proteins‐Protein folding
The Endoplasmic Reticulumis the gateway for proteintransport into all the othermembrane ‐bound organellesof the secretory pathway.
Proteins and lipids are trafficked through the secretory pathwayin small carriers called vesicles.
The Secretory Pathway
lysosome
Transport from the ER through the Golgi Apparatus
Proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into small COPII‐coated transport vesicles. These transport vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes.
The Steps of Vesicle Transport
After transport vesicles have budded from an ER exit site and have shed their coat, they begin to fuse with one another forming vesicular tubular clusters.
Membrane fusion requires a set of matching SNAREs [SNAP (Soluble NSF Attachment Protein) Receptor]
Vesicle Fusion requires SNARE proteins
Once the vesicular tubular clusters are form, they begin to budding off resulting into new vesicles, these vesicles are COPI‐coated. They carry back ER resident proteins that have escaped as well as proteins that participated in the ER budding reaction.
Formation of a transport vesicle requires coat proteins
Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face (or exit face).
Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golginetwork (CGN) (also called the intermediate compartment) and the trans Golgi network (TGN), respectively.
Proteins exiting from the TGN can either move onward and be sorted according to whether they are destined for lysosomes, secretory vesicles, or the cell surface.
Transport from the Trans GolgiNetwork to Lysosomes
Lysosomes are membrane‐enclosed compartments filled with hydrolytic enzymes that are used for the controlled intracellular digestion of macromolecules.
All are acid hydrolases. For optimal activity they require an acid environment, and the lysosomeprovides this by maintaining a pH of about 5.0 in its interior. An H+
pump in the lysosomal membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH.
Newly synthesized lysosomal proteins are transferred into the lumen of the ER, transported through the Golgiapparatus, and then carried from the trans Golgi network to late endosomes by means of clathrin‐coated transport vesicles.
The lysosomal hydrolases contain N‐linked oligosaccharides that are covalently modified in a unique way in the cis Golginetwork
These mannose 6‐phosphate (M6P) groups are recognized by an M6P receptor protein in the trans Golgi network that segregates the hydrolases and helps package them into budding transport vesicles that deliver their contents to late endosomes. The M6P receptors shuttle back and forth between the trans Golgi network and these endosomes.
Transport into the Cell from the Plasma Membrane: Endocytosis
Endocytosis is a process in which the particle move inward from the cell surface to lysosomes.
Two main types of endocytosis are distinguished on the basis of the size of the endocytic vesicles formed.
Phagocytosis (“cellular eating”), which involves the ingestion of large particles, such as microorganisms or dead cells via large vesicles called phagosomes (generally >250 nm in diameter). It is mainly carried out by specialized cells —macrophages, neutrophils, and dendritic cells.
Pinocytosis(“cellular drinking”), which involves the ingestion of fluid and solutes via small pinocytic vesicles (about 100 nm in diameter). Pinocytic vesicles often begins at clathrin‐coated pits in the plasma membrane
lysosome
Most eukaryotic cells are continually ingesting fluid and solutes by pinocytosis; large particles are most efficiently ingested by specialized phagocytic cells. Pinocytosis, which is a constitutive process that occurs continuously.