3.5.2. Mitochondrial targeting :
Mitochondria and chloroplast are double layered membrane organelles and contain internal subcompartments. Both organelles also contain similar type of electron transport proteins and use an F-class ATPase to synthesize ATP (also in gram negative bacteria).As bacteria , mitochondria and chloroplast also contain their own DNA, rRNA, tRNA and some proteins so growth of these organelles does not depend on nucleus. Their growth depends on incorporation of cellular proteins and lipids and on division of pre-existing organelles (both processes occurs in interphase of cell cycle).
Due to numerous similarity between bacteria and these organelles, this is supposed to called endo-symbiotic organelles, as evidence found for the ancient evolutionary relationship in some proteins. But an unknown mechanism is found that tells –some precursor proteins synthesized in cytosol are targeted into matrix of mitochondria, the stroma of chloroplast , which usually contain specific N-terminal uptake targeting sequences that specifies for the receptor proteins on the organelle surface, which cleaves, removes later and protein enters inside organelle.
This N-terminal sequence is basically amphipathic helix, 20-50 residues in length with ser, thr, arg, lys positively charged and hydroxylated amino acids / residues on one side and hydrophobic residues on the other. This sequence lack negatively charged amino acids (asp and glu).
Mitochondrial protein import requires outer membrane receptors and translocon in both membranes:
The soluble precursors of mitochondrial protein (including hydrophobic integral membrane protein ) after synthesis in the cytosol, interact directly with mitochondrial membrane (in general, only unfolded protein can be imported into mitochondria by cytosolic chaperone protein Hsc70).
Mitochondrial targeting sequence (MTS) is present on mitochondrial protein/precursor binds to an import receptor in the outer mitochondrial membrane (OMM).
Specific receptor protein are responsible for import of different class of mitochondrial protein.(eg.-N-termianl MTS are recognized by TOM20 and TOM22) [proteins in OMM involved in targeting and import are designated TOM proteins for translocon of the OM].
The import receptor subsequently by unidirectional transport the targeted protein to an import channel in the outer membrane. (Import channel such as, - Tom40 protein, is known as General import pore because all imported protein transport through this transmembrane channel to the interior compartments of mitochondrion).
3.5.3. Protein import into the mitochondrial matrix
In case of precursor protein destined into matrix transfer through an inner membrane channel (eg-Tim23/17 proteins). Tim represents Translocon of inner membrane. Translocation into the matrix that occurs at contact sites, where the outer membrane and inner membrane are in close proximity. After entering in matrix, N-MTS is removed by a protease, that resides in matrix. Precursor protein is also bound by matrix Hsc70, a chaperone that is localized to translocation channel in IMM by Tim44 interaction and together these protein interaction stimulates ATP hydrolysis by Hsc70,which provides power to translocate protein into matrix. As we know, final folding of many protein requires a chaperonin, otherwise assembly of protein fails in folding.
Three energy inputs are needed to import proteins into mitochondria:
- ATP hydrolysis by Hsc70 chaperone proteins in both cytosol and the mitochondrial matrix is required for import of mitochondrial proteins (invivo only)
- The matrix Hsc70, anchored to membrane by Tim44 protein, may act as a molecular motor to pull the protein into the matrix. (Hsc70 & Tim44 would be analogous to the chaperone BiP and Sec63 complex respectively, in post translocation into ER lumen).
- The third energy input required for mitochondrial protein import is a proton electrochemical gradient or proton motive force, across the inner membrane i.e. only respiration undergoing mitochondria are able to translocate precursor protein from cytosol into mitochondrial matrix.
3.5.4. Multiple signals and pathways target proteins to sub- mitochondrial compartments:
Three pathways for transporting proteins from cytosol to inner mitochondrial membrane
- One pathway make use of the some machinery used for targeting of matrix proteins. A cytochrome oxidase subunit (CoxVa) is a typical protein transported by this pathway (involvement of Tim23/17 channel).
- A second pathway to the inner membrane is followed by proteins (ex-ATP synthase subunit 9) whose precursors contains both a matrix targeting sequence and internal hydrophobic domains recognized by an inner membrane protein termed oxa1, (involvement of Tim23/17 and Tom 20/22 channels).
- The third pathway for insertion in the inner mitochondrial membrane is followed by multipass proteins that contain six-membrane spanning domains, such as ADP/ATP antiport lacking usual N-terminal matrix targeting sequence, contain multiple internal mitochondrial targeting sequence.
Chloroplast word is derived from Greek words Chloros-"green" & plastes-"the one who turns". Chloroplast are specialized cell organelles occurs in all photosynthetic eukaryotes plants and algal cells. Discovered by Julius Von Sachs (1832-1897) on influential botanist and author of standard botanical textbooks, sometimes called "The father of plant physiology". A Chloroplast is one of three types of plastids, characterized by having high concentration of chlorophyll, the other two types, the leucoplast & the chromoplast, contain little chlorophyll and do not carry out photosynthesis. Chloroplast are double-membrane bound semi autonomous organelles, that conduct photosynthesis, where the chlorophyll (photosynthetic pigment) capture the energy from sunlight & convents it and stores it in the energy storage moleculs ATP & NAPH while hydrolysis of water.
They then use ATP and NAPH for CO2 fixation in a process known as tricarboxilic acid (TCA) cycle. Fatty acid Synthesis, some amino acid synthesis and the immune response in plant conclucded by chloroplast. Its number are varies from 1 in algae up to 100 in plants like Arabidopsis & wheat. (abbreviated as ctDNA or cpDNA it is also known as plastome).
Chloroplasts, like mitochondria, contain their own circular DNA which is thought to be inherited from their ancestor a photosynthetic cyanobacterium that was engulfed by an early eukaryotic cell. Chloroplast can't be made by the plant cell and must be inherited by each daughter cell during cell division like mitochondria, the chloroplast also divide by binary fission this is strongly influenced by environmental factors like light, colour and intensity.
Chloroplast are considered to be originated from aerobic cyanobacteria through endosymbiosis by eukaryotic cell, (blue-green algae photosynthesizing) that become a permanent resident in the cell. The origin of chloroplast was 1st suggested by Russian biologist konstantin Mereschkowski in 1905. Chloroplast are generally lens -Shaped, 5-8 mm in diameter and 1-3 mm thick.
All chloroplast have at least three membrane systems.
- The outer chloroplast membrane.
- The inner chloroplast membrane.
- The thylakoid system.
Inside the outer and inner chloroplast membranes is the chloroplast stroma a semigel-like fluid that makes up much of the chloroplast's volume, in which thylakoid system floats.
The outer Chloroplast membrane is a semi-porous membrane, through which small molecules and lens can easily diffuse across. However it is not permeable to larger proteins, so chloroplast polypeptide being synthesized in the cell cytoplasm must be transported across the outer chloroplast membrane by TOC complex, or translocon on the outer chloroplast membrane. Chloroplast double membrane which encloses a fluid filled region called the stroma.
The protein rich, alkaline, aqueous fluid, which corresponds to the cytosol of the original cyanobacterium. Nucleoids of chloroplast DNA, Chloroplast ribosome, starch granules, many proteins and plastoglobuli can be found floating around in it. The CO2 fixation into starch (sugar) i.e. Calvin cycle takes place in the stroma.
Intermembrane space and peptidoglycan wall
- About 10-20 mm wide, thin intermembrane space exists between the outer and inner chloroplast membranes. Some algal (Glaucophyte) chloroplasts have a peptidoglycan layer between the chloroplast membranes, which corresponds to the peptidoglycan cell wall of their cyanobacterial ancestors, which is located between their two cell membrane. These chloroplasts are called muroplasts (latin-"Muro" meaning "Wall". In may chloroplast the cyanobacterial wall is lost, leaving an intermembrane space between the two chloroplast envelope membranes.
- Inner chloroplast membrane-borders the stroma and regulates passage of materials in and out of the chloroplast. The inner membrane has TIC complex, translocon, through which the polypeptide must pass. The inner chloroplast membrane are the site for fatty acids, lipids & carotenoicls are synthesized.
- The thylakoid system : Greek word thylakoids which means "sack". A highly dynamic collection of membranous sacks, suspended within the chloroplast stroma called thylakoids, Where chlorophyll is found & the light Reactions of photosynthesis happen. In most vascular plant chloroplasts, the thylakoids are arranged in stacks called grana, where as some C4 plants and some algal chloroplast, the thylakoids are free floating.
Each stack of flattened sac of thylakoid is made up of the grana, and the other long interconnecting stromal thylakoids which linked different grana. Thylakoid membrane have imp. protein complex which carry out the light reactions of photosynthesis. PS I and PS II contain light-harvesting complex with chlorophyll and carotenoids that absorb light energy & used it to energize es. Molecules in the thylakoid membrane use the energized es to pump H+ into the thylakoid space, decreasing the pH and turning it acidic.
ATP is generated as the H+ ions flow back out into the stroma.Chloroplast stromal proteins are the enzymes of calvin cycle ,which functions in fixing carbon dioxide into carbohydrates during photosynthesis.
The larger (L) subunit of ribulose 1,5 bisphosphate carboxylase (RUBISCO) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes in the stromal space. The small (S) subunit of rubisco and all other calvin cycle enzymes are encoded by nuclear genes and transported to chloroplast after their synthesis in cytosol. The precursor forms of these precursor stromal proteins (eg-S subunit) contain an N-terminal stromal- import sequence (SIS). (No common motifs, generally rich in ser, thr and small hydrophobic residues and poor in glu and Asp).These proteins are reached in an unfolded state in stroma, by binding transiently to a stromal Hsp70 chaperone and later N-SIS is removed. Eight (S) subunit combine with eight L-subunit to yield the active rubisco enzyme by facilitating reaction of Hsc60 chaperonins.
Although chloroplast are functionally analogous to the receptor and channel proteins in mitochondrial membrane, so they are not structurally homologous. The lack of homology between these organelles suggests that they may have arisen independently during evolution.
Import into the stroma depend on ATP hydrolysis catalyzed by a stromal function is similar to Hsp70 in mitochondrial matrix and BiP in ER lumen. But chloroplast can not generate an electrochemical gradient (PMF) across their inner membrane,unlike mitochondria.Thus protein import into chloroplast stroma appears to be powered solely by ATP hydrolysis.
Proteins are targeted to thylakoids by mechanisms related to translocation across the bacterial innermembrane: Proteins destined for thylakoid have secondary targeting sequences.After entry of these proteins into the stroma, cleavage of the stromal targeting sequence reveals the thylakoid targeting sequence.The two pathways (Figure-8) for moving protein from stroma to thylakoid resemble translocation across the bacterial inner membrane. One of these systems can translocate folded proteins.
3.7. Peroxisome Targeting :
Peroxisomes are single membrane bound small organelles (0.5 - 1mm in diameter) found in nearly all eucaryotic cells. Their existance was 1st discovered by J. Rhodin in 1954 and they were officially consider organelles in 1967 by christion de Duve. Theses organelles mainly occurs in photosynthetic cells of higher plants, algae, liverworts, mosses, ferns and also in fungi. Their number varies from 70-100 per cell. Peroxisomes are rounded bodies whose diameter varies from 0.2-1.5 m. It was believed that peroxisome evolved from bacteria by endosymbiotant, that formed a symbiotic relationship with their host cell. It was believed that the development of this relationship over generation leads to bact evolving as an organelle inside the body. Peroxisomes resemble organelles found in other organisms as they are related to glyoxysomes of plants fungi and also glycosome of kinetoplastids.
Peroxisomes are membrane-bound organelles found in both animal and plant cells that contains and abundance of enzyme for detoxifixing harmful substances and lipid metabolism. That derived from the ER and replicate by fission. This organelle is surrounded by a lipid bilayer membrane which encloses the Crystalloid core. The bilayer is enclosed with plasma membrane which regulates what enters and exits the peroxisome. Peroxisomal matrix proteins are translated in the cytoplasm prior to import. There are at least 32 known peroxisomal proteins, called peroxins which carry out peroxisomal function inside the organelle. The matrix of peroxisome contains peroxide-destroying enzymes (catalases) and peroxide producing enzymes. They prevent the peroxides from acting on the cellular contents.
Lipid metabolism and chemical detoxification are imp fn of peroxisiomes.
Peroxisomes are responsible for oxidation reactions that break down fatty acids and amino-acids.
Peroxisomes oversee reactions that neutralize free radicals, which cause cellular damage and cell death.
Peroxixomes chemically neutralize poisons through a process that produces large amount of toxic H2O2, which is then converted into H2O and oxygen.
The liver is the organ primarily responsible for detoxifying the blood before it travels throughout the body; as a result, liver cells contain large amount of peroxisomes.
Peroxisome are small organelles bound by a single membrane. Unlike mitochondria and chloroplast ,peroxisome lack DNA and ribosomes. So, all peroxisomal proteins are encoded by nuclear genes,synthesized on ribosomes free in the cytosol and then incorporated into pre-existing or newly generated peroxisomes. Peroxisomes are abundantly in liver cells. Most peroxisomal matrix proteins contain a C-terminal PTS1 targeting sequence; a few have an N-terminal PTS2 targeting sequence. Neither targeting sequence is cleaved after import. All proteins destined for peroxisomal matrix bind to a cytosolic receptor, which differs for PTS1 and PTS2 bearing proteins and then are destined to common import receptor and translocation machinery on the peroxisomal membrane.
Zell-weger syndrome is the disorder in the defect of peroxisome assembly, an autosomal recessive mutation, that occur naturally in human population. In this transport of many proteins into peroxisomal matrix is impaired, newly remain in cytosol or eventually degraded.
Perspective of the future:
A more detailed understanding of all translocation processes should continue to emerge from genetic and biochemical studies, both in yeast and in mammals.
The nucleus is the double membrane occupies about 10% of the total cell volume bound, the largest cellular organelle and the controlling center of eukaryotic animal cell. Cell nuclei contain most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a protein (Histone), to form chromosome. Eukaryotes usually have single nucleus, but a few cell types, such as mammalian red blood cells have no nuclei and a few other have many.
Paramecia-have two nuclei-macro and micro nucleus. The nuclear envelope is made up of a double membrane st that provides a barrier between the nuclear contents and the cytosol-the inner nuclear membrane and outer nuclear membrane. They are connected together, but their protein compositions are different. The inner nuclear membrane contains integral and peripheral membrane proteins that anchor the nuclear envelope to the lamina, which is a sturdy protein meshwork that gives the nucleus its structure and shape.
The outer nuclear membrane is contiguous with the ER, which is the intracellular compartment where lipids, as well as proteins that are going to be secreated or inserted into membrane are much. The ER and outer nuclear membrane both are studded with ribosomes, which are the enzymes that translate mRNA into protein. The space between the inner and outer nuclear membrane is called perinuclear space, it is continuous with the inside of the ER, so the same processes occur in the ER as in the perinuclear space. Although the nucleus is a separate compartment from the cytosol, many molecules have to go in a out, these included histone, RNA, DNA, ribosome, Polymerase, to factors etc. through nuclear pore.
3.8.1. Nuclear targeting :
Proteins are not transported through the nuclear membrane but rather through a complex pore called the nuclear pore, which is comprised of-
- About 100 different proteins
- Proteins smaller than 20 KDa by selective transport
- Proteins larger than 20KDa by selective transport (nuclear localization signal; NLS), which is a cluster of 4-8 positively charged amino acids (eg- PKKKRLV)
Usually, nucleus targeted proteins follows two way traffic: in and out
In: Nucleoplasm involves proteins, DNA.
Inside nucleus, DNA and RNA polymerases, transcription factors, histones etc. are targeted across the nuclear pore.
Out: Outside nucleus, mRNA, tRNA, rRNA are generally targeted across the nuclear pore.
Proteins are targeted to the nucleus by a specific amino acids sequence as phenylalanine glycine repeats (FG repeats) while some proteins exits from nuclear requires a nuclear export sequences (NES). Nuclear import & export pathways are mediated by a family of soluble receptor referred to as importin and exportin and collectively called karyopherins (alpha/importin beta1 heterodimer; designated as a and b). The best studied NLS are basic amino acids sequence typically rich in Lys and Arg.
The best characterized nuclear transport sequence are the small hydrophobic leucine rich nuclear export sequences, 1st described in HIV Rev protein ( Crm-1 exportin).An importin binds to its NLS nearing cargo/protein in the cytoplasm and translocates through the NPC into the nucleus. The importin binds Ran–GTP in the nucleus, resulting in cargo varies. The importin–Ran–GTP complex recycles back into the cytosol after translocation to the cytoplasm, GTP hydrolysis on Ran by Ran–GAP.
A gradient of Ran–GTP exists in the cell with Ran–GDP add high concentration in the cytosol and Ran–GTP at a high concentration in the nucleus to maintain the high nuclear concentration of Ran, a dedicated transporter protein NTF2 functions to recycle Ran–GDP continuously back to the nucleus. Ran–GTP is generated in the nucleus by chromatin bound Ran–GEF.
Cargo protein (protein which needs to transport from cytoplasm to nucleus) for importin present in cytoplasm. Impotin get responsible to transfer it respective cargo from cytoplasm to nucleus, to accomplish this function binding between cargo and importin takes place, in turn they become capable to make interaction with nuclear pore and as a result pass from its cannel and comes inside nucleus. In nucleus Ran–GTP complex binds to importin and cargo complex which cause conformational change in importin, in turn, importin dissociation with its cargo takes place, as a result cargo get transported to cytoplasm and now translocation of Ran–GTP complex along with importin from the nucleus to the cytoplasm takes place. In cytoplasm Ran binding protein (Ran BP) binds to Ran and cause the separation of Ran–GTP complex from importin, as a result a access generate for GTPase activating protein (GAP) to get bind to Ran–GTP complex, in turn cause hydrolysis of GTP occur and formation of Ran–GDP takes place. Ran–GDP binds to nuclear transport factor which mediate its transfer from cytoplasm to nucleus. In nucleus Ran–GDP complex trigger by guanine nucleotide exchange factor which convert GDP into GTP and formation of Ran–GTP complex takes place, now this complex again start new cycle.
In nucleus exportin binds to its respective cargo protein, then this complex binds with Ran–GTP complex and along with Ran–GTP complex, exportin and its cargo get diffuse from the nuclear pore and reach into the cytoplasm. In cytoplasm GAP protein binds to Ran–GTP complex and causes hydrolysis of GTP in turn cargo get release in nucleus. After that Ran–GDP move into the nucleus with a ligand which it takes from cytoplasm and in nucleus it again get converted in to Ran–GTP by the action of GEF and release is ligand in nucleus.