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LET'S TALK LIFE-SCIENCE CELL BIOLOGY

Suraj Prakash Sharma | Ekta Chotia

PROTEIN TARGETING
207

3.1.1.     Endoplasmic reticulum (ER) :

First reported by Porter in 1945. The Endoplasmic reticulum (ER) is an organelle found in the cells of eukaryotic organisms. It is an extensive interconnected network of closed and fattened tube like membrane bound structure throughout the cell. These membranes are continuous, joining with the outer membrane of the nuclear membrane. On one end and cell membrane on there other end. ER occurs in almost every type of eucaryotic cell except red blood cells and sperm cells. The ER functions as a manufacturing and packing system. It works closely with the golgi apparatus, ribosomes, mRNA and tRNA.

The membranes are slightly different from cell to cell. A cell's function determines the size and structure of the ER for eg. some cells such as prokaryotes or red blood cells do not have an ER of any hand. Cells that synthesize and release a lot of proteins would need a large size of ER such as cells of liver and pancreas.

The ER contain three different types of structure. These are 1) Cisternae , 2) Vesicles and 3) Tubules.

  1. Cisternae : These are network of long, flat and unbranched membrane plates or lamellae arranged in parallel rows, having sac like structure which are held together by the cytoskeleton. The phospholipid membrane encloses the cisternal space (lumen) which is continuous with the peri nuclear space but separate from the cytosol.
  2. Vesicles : They are usually round or ovoid sacs. They often occur isolated in the cytoplasm and they transport ER's proteins cargo to the golgi complex for distribution.
  3. Tubules : They are irregularly branched tube-like structure having a diameter of 50-100 n. These are surrounded by this unit membrane of 50-60 thickness and their lumen is filled with the secretary products of the cell.

There are 2 Types of E.R.

  • Smooth Endoplasmic Reticulum (SER) : The surface of this type of reticulum is smooth as ribosome's not attached to it. Smooth ER is actively engaged in.
  1. Steroid synthesis like-cholesterol, progesterone, testosterone etc.
  2.  Lipid synthesis.
  3. Glycogen synthesis.
  4. Metabolism of carbohydrates.
  5. Detoxification function.
  6. Major storage and released site of inter cellular calcium ions.
  • Rough Endoplasmic Reticulum (RER) : Rough Endoplasmic have ribosome attached throughout the surface. These are present in cells, which are active in.
  1. protein synthesis.
  2. Protein translocation, folding and transport of protein.
  3. Glycosylation (this is the relation of saccharide group with a hydroxyl or amino functional group to form a glucoside).
  4. Disulfide bond formation (d-S-S bonds stabilize the tertiary and quaternary structures of many proteins).
  5. Membrane synthesis.

Common functions of Both ER (SER and RER) are :

  1. Form the skeletal framework.
  2. Active transport of cellular materials.
  3. Metabolic activities due to presence of different enzymes.
  4. Provide increased surface area for cellular reactions.
  5. Formation of nuclear membrane during cell division.

Microsomes - are typically vesicles of fragmented endoplasmic reticulum to which ribosomes are attached, called rough microsome and on which ribosomes are not attached one called smooth microsomes.

3.1.2.     Protein sorting to ER :

General sorting process applies to proteins that initially are targeted to the ER membrane, entering the Secretory Pathway. These protein include not only soluble and membrane proteins that reside in the ER itself but also proteins that are secreted from the cell, enzymes and other resident proteins in the lumen of golgi complex and lysosome and integral proteins in the membranes of these organelles and the plasma membrane. These proteins are transported along the secretory pathway by small vesicles that bud from the membrane of the organalle and then fuse with the membrane of the next organelle in the pathway. The signal sequence hypothesis was first enunciated by Gunther Blobel who was awarded the Nobel Prize in medicine in 1999 for his work.

Despite some variations, the same basic mechanisms govern protein sorting to all the various intracellular organelles (containing signal sequences or uptake targeting sequences). Each organelle carry a set of receptor proteins that bind only to specific kinds of signal sequence that slows specificity of targeting .Once a protein containing a signal sequence interact with specific receptor, the protein chain is transferred to a translocation channel that allows protein unidirectionaly transfer to pass through the membrane bilayer, achieved by favourable process such as hydrolysis of ATP. Finally, signal sequence are removed from mature protein by specific protease, once translocation is completed.

3.1.3.     Translocation of secretory proteins across the ER membrane:

Free and membrane bound ribosomes are functionally distinguishable and all proteins synthesis initiate on ribosome that are free in cytosol. Ribosome engaged with synthesis of protein that are destined part of secretion are then entry to the ER by a signal sequence at the N-terminal of the growing polypeptide chain.

These signal sequence short stretches of 6-12 hydrophobic amino acid that are usually cleaved from polypeptide chain into the ER lumen by N-signal peptidase.

Some transport of most secretory protein into ER lumen occurs while the nascent protein is still bounded to the ribosome and being elongated, a process referred to as co translational modifications.

Signal hypothesis:

As the nascent protein is emerged from ribosome, signal sequence is recognized and bound by Signal Recognition particle (SRP) a cytosolic ribonucleoprotein practicle. Six discrete polypeptide and 300 nucleotide RNA constitute the SRP.

SRP contains a small, cytoplasmic RNA. The SRP RNA binds with the ribosome as well as the targeting signal sequence, inhibiting further translation targeting entire complex translation Rough ER by binding to SRP receptor on the ER membrane. Two of the SRP proteins,P9 and P14 interact with ribosome while P68 and P72 are required for protein translocation. SRP wraps around the large ribosomal subunits with one end binding to the ER signal sequences and the other end block the elongation factor binding site. This halts the translation. The ribosome binds to protein at translocation complex, signal sequence is inserted into a protein channel, sometimes called translocons (Sec61 complex comprising á,â,ã subunits are found to form mammalian translocons).

Entire process is coordinated by GTP binding to both SRP at SRP receptor. With hydrolysis of GTP, leading to dissociation of SRP from both the receptor and ribosome mRNA complex. Transfer of ribosome on mRNA complex from SRP to the translocon opens the gate on the translocon allows translocation to resume and the growing polypeptide chain is transferred directly into translocon channel across ER membrane. The signal sequence is cleaved by N-signal peptidase and polypeptide is released into the lumen of ER. ATP hydrolysis powers post-translational translocation of some secretory proteins in yeast:

In most eukaryotes, secretory protein enter by cotranslational translocation using energy destined from translation to pass through the membrane as we have just described. In yeast, SRP and SRP translation translocation are not involved in post translational translocation as in such cases a direct interaction between the translocation and signal sequences of completed protein appears to be sufficient for targeting the ER membrane.

In this case, an additional protein in ER membrane (is known as Sec63 complex and a member of Hsc family of chaperonins known as BiP) are provided for unidirectional translocation. BiP contains a peptide binding and an ATPase domains . These chaperones bind and stabilize unfolded or partially folded proteins.

3.1.4.     Insertion of proteins into ER membrane:

Each protein has a unique orientation with respect to membrane’s phospholipid bilayer. During transport, the orientation of a membrane protein is preserved i.e. the same segment of the protein is always face the cytosol while other segments always face in the opposite direction. Examination of several types of sequences collectively known as topogenic sequences, direct the insertion and various classes of integral proteins into the membrane. Several topological classes of integral membrane proteins are synthesized on ER. Four topological classes are illustrated as to categorize integral membrane proteins.

Topological classes I, II & III comprise single pass proteins, which have only one membrane spanning a-helical segment. The proteins forming topological class- IV contain multiple membrane spanning segments.

Synthesis and insertion into ER membrane of type I single pass protein. The type one polypeptide has signal sequence followed by the long hydrophobic patch. This hydrophobic patch is inserted into the membrane of ER. 

Synthesis and insertion into ER membrane of type II single pass protein. The type 2 polypeptide has a hydrophilic patch followed by the signal sequence. This hydrophilic patch does not allow the insertion of this polypeptide into the ER membrane thus it remain in the cytosol.

The topology of a membrane protein often can be deduced from its sequence. Identification of topogenic sequences requires a way to scan sequences databases for segments that are sufficiently hydrophobic to the either a signal sequence or a transmembrane anchor sequence.Topogenic sequence can often be identified with the aid of computer programs that generate a Hydropathy Profile for the protein of interest.

Hydropathy plot of connexin

The hydropathy index on the vertical axis is a numerical measure of the relative hydrophobicity of successive segments of the polypeptide chain based on its amino acid sequence. Hydropathy Analysis of an Integral Membrane Protein. A hydropathy plot is a means of representing hydrophobic regions (positive values) and hydro  philic regions (negative values) along the length of a protein. The prominent peaks in such plots identify probable topogenic sequences as well as their position and approximate length.

Transmembrane structure of connexin. Connexin has four distinct hydrophobic regions, which correspond to the four α-helical segments that span the plasma membrane.

Some cell surface proteins are initially synthesized as type I protein on the ER and then cleave with their luminal domain transferred tp a GPI anchor.

3.1.5.     Protein modifications,folding and quality control in ER:

For secretory proteins, the ER is the site of protein folding assembly of multisubunit protein, disulfide bond formation, initial stages of glycosylation and addition of glycolipid anchors to some PM proteins.

Disulfide bond formation:

This is facilitated by the enzyme ,Protein disulfide isomerase (PDI) located in ER lumen.

PDI contains an active site with two closely spaced cysteine residues that are easily interconverted between the reduced dithiol form and the oxidized disulfide form. In the formation of disulfide bonds, the ionized (–S–) form of a cysteine thiol in the substrate protein reacts with the disulfide (S-S) bond in oxidized PDI to form a disulfide-bonded PDI–substrate protein intermediate. A second ionized thiol in the substrate then reacts with this intermediate, forming a disulfide bond within the substrate protein and releasing reduced PDI.

Reduced PDI can catalyze rearrangement of improperly formed disulfide bonds by similar thiol-disulfide transfer reactions. In this case, reduced PDI both initiates and is regenerated in the reaction pathway. These reactions are repeated until the most stable conformation of the protein is achieved.

3.1.6.     N-Linked Glycosylation:

Proteins are glycosylated at specific N-residue within the ER, while their translocation is still in process. Such type of glycosylation is called N-LINKED glycosylation.More than 90% glycosylation in protein is N-linked.

N-linked oligosaccharide consists of 14 sugars (2 NAG, 3Glucose, 9Mannose) units that are added to receptor Asn (N) residue to growing polypeptide chain, as they are translocated into ER. The oligosaccharide assembled in ER is transferred as an intact unit (complete 14 sugars) to N-residues.The precursor oligosaccharide is linked to dolichol lipid by a high energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction.

The enzyme which transfers oligosaccharides moiety from dolichol-Phosphate to N-residue is oligosachharyl transferase (OST). One copy of this enzyme is associated with each protein translocator in ER membrane.

3.2.         Golgi apparatus:

The Golgi apparatus, also known as the Golgi complex, Golgi body or simply the Golgi in an cells organelle found in most Eucaryotic Cells. It was first discovered by the Italian physician Camillo Golgi in 1897 & named after him in 1898. The golgi apparatus is the "manufacturing and shipping center"of Eucaryotic cell. In mammals, and single golgi complex or apparatus is usually located near the cell nucleus, close to the centrosome.

Each stack of golgi are linked together through tubular connection which depends on microtubules. If microtubles are experimentally depolarized, then golgi apparatus loses connections and become individuals stacks throughout the cytoplasm.

In, yeast, multiple golgi apparatuses are scattered throughout the cytoplasm. In plants golgi stacks are not concentrated at the centrosomal region and do not form golgi ribbons. Organization of the plant Golgi depends on actin cables and not microtubules.

A golgi apparatus is composed of flat sacs known as cisternae. The sacs are tacked in a bent semicircular shape. Each stacked grouping has a membrane that separates it's insides from the cell's cytoplasm. Golgi membrane protein interactions are responsible for its unique shape. These interactions generate the force that shape this organalle. The golgi apparatus is very polar. Membrane at one end of the stack differ in both composition and in thickness from those at the other end. One end (Cis face) act as "receiving" department while the other (trans face) acts as the "shipping" department. The Cis face is closely associated with the ER.

The Golgi apparatus is made up of a series of compartment of single membrane bound organelle and part of endomembrane system. It consist of two main networks i) Cis Golgi network (CGN), II) trans Golgi network (TGN). The CGN is a collection of fused, flattened membrane enclosed disks known as cisternae, originating from vericular clusters that bud off from the endoplasmic reticulum.A mammalion cell typically contain 40 to 100 stacks. In some protists as many as cisternal have been observed. The three major cisternae compartment are Cis, median & trans compartments. The CGN has its face also called entry face or forming face. The TGN is the final cisternal structure, from which proteins are packaged into vesicles destined to lysosomes, secretory vesicles or the cell surface. The trans face of TGN alro called maturing face TGN was earlier known as GERL (Golgi-apparatus-ER-lysosome).

It is a major site of carbohydrate synthesis as well as final sorting and dispatching centre for products of the endoplasmic reticulum. While in endoplasmic reticulum, many proteins undergo the first stage of glycosylation. Most proteins then migrate inside vesicle from ER and enter the cis-face of the golgi. Golgi membrane are studied with glycosyl transferase, glycosidases and nucleotide sugar transporter arranged in a generally ordered manner from the cis-golgi to the trans-golgi network. (TGN), such that each activity is able to act on specific substrate generated earlier in the pathway.

A large portion of the carbohydrate, that makes are attached on oligosaccharide side chains to the many proteins and lipids that the ER membrane to it. A subset of these oligosaccharide groups serve as tags to direct specific protein into vesicle that then transport them to lysosome. Golgi apparatus consists of a collection of flattened membrane include compartments called cisternae.

During their process to golgi apparatus, transported molecules undergo and ordered series of covalent modifications. Complex oligosaccharides are generated, when the original N-linked oligosaccharides added in endoplasmic reticulum is trimmed and further sugar are added.

High mannose oligosaccharide are trimmed but has no new sugars added to them in the golgi apparatus. Complex oligosaccharide can contain more than the original 2 NAGs as well as available number of galactose, sialic acid and in some cases fucose. Sialic acid is not found in plant system.

3.2.1.     O-linked glycosylation:

Glycosylation can also be processed at Serine, Threonine and Tyrosine residues. It is called O-LINKED GLYCOSYLATION. These are also added within the golgi apparatus while the sequential addition of single sugar residue. The S/T are usually linked directly to NAG to which other sugars can be added. Many cytoplasmic and nuclear protein including a variety of transcription factors are also modified by single O-linked NAG residue, but this transcription process is reversible. In some cases, o-linked glycosylated sugars are further modified by addition of sulfate sugars. Monoglycosylation by O-NAG occurs only on cytoplasmic and nuclear proteins. The site of monoglycosyaltion are often identical to the site of phosphorylation.

Eg- The transcription regulator, c-myc (in cell division) contains Threonine residues, i.e. either glycosylated or phosphorylated. The carboxy terminal domain (CTD in RNA Polymerase II) becomes phosphorylate at S-2 and S-5 of the heptapeptide repeats (YSPTSPS) in the cores of transcription cycle. The CTD can also be monoglycosyalated with NAG at position 4 (Thr) of the heptapeptide. Ex- there is an evidence that monoglycosylation play a role in nuclear localization and in the modulation of protein protein interaction.

Conclusion:

The nature of glycan attached to any given protein varies from species to species and often from one tissue to next. Protein glycosylation is almost exclusively a eukaryotic property and the complexity of modification increases as one proceeds up the evolutionary tree.

Lower eukaryotes like yeast attach only a simple set of sugars to their proteins, whereas mammals modify their protein with highly branched oligosaccharide composed of a wide range of carbohydrates.

The N-linked glycosylation are usually more complex than O-linked in nature. The importance of these glycosylation steps in human development as well understood by finding that mutations of any of the roughly 15 genes encoding proteins involved in N-glycosylation result in a variety of developmental disorders, including mental retardation and motor deficits.

3.2.2.     Significance of glycosylation:

Protein glycosylation serves as a variety of functions

  1. The attachment of glycan generally increases the solubility of nascent glycoprotein inside the cell and can prevent their aggregation.
  2. Small monosaccharide attached to proteins within the cell also are thought to find role in intracellular signaling.
  3. One of the protein is secreted, glycosylation can provide protection from protease and from non-specific protein-protein interaction.
  4. Glycosylated protein on the cell surface can also be bound by other proteins surrounding the cells that recognize the specific interaction.This type of interaction commonly plays role in recognition by one cell by another (cell-cell interaction)
  5. The oligosaccharide intermediate created by the trimming reaction help in protein folding and transport of misfolded protein to cytosol for degradation. Thus, they plays an important role in controlling the quality of protein.
  6. Various type of antigen present on RBC plasma membrane are also example of glycosylation.

3.3.         Vesicle transport:

Most important vesicles that carry secretory protein from ER to golgi and from golgi to other targets are coated with cytosolic coat protein and thus are called coated vesicles.

The budding of vesicles from ER to GOLGI is assisted by COP-II.After budding out of ER ,the vesicles loose their COP II coat and fuse together to form the tubular clusters. This fusion is promoted by the v-SNARE & t-SNARE of the vesicles.

While vesicles moving from GOLGI to ER (retrival transport) utilized COP-I.Retrieval signals are usually located at the C-terminal end of the protein. For ER membrane proteins, it is usually KKXX (Lys-Lys-X-X)-This sequence is present on KDEL receptors (multipass transmembrane protein) to bind the COPI coat.

For soluble ER resident proteins,it is usually KDEL(Lys-Asp-Glu-Leu or something similar).

KDEL receptors have high affinity for KDEL tubular clusters and golgi,while KDEL receptor have low affinity for KDEL in ER (including its release in ER).

There is a pH gradient from ER (neutral) to golgi (acidic).This pH difference is regulated by V-type ATPases (proton pumps).The pH effect plays a crucial role in KDEL receptor binding:

  1. Low pH, high affinity
  2. Neutal pH, low affinity

But not all proteins have retrieval signals.

A third type of COP protein clathrin is required for budding of vesicles from PM and possible from the trans-golgi.Clathrin coated vesicles are the best understood and are responsible for uptake of extracellular molecules from PM by endocytosis as well as transport of molecules from TGN to endosome, lysosome or to the PM. Thus, clathrin coated vesicles participitate in endocytic recycling ,retrograde transport the bring soluble and membrane bound proteins into the cell. Before a vesicle fuse, with a target membrane,  discard its coat.

Coat recruitment GTPase control the assembly of clathrin coat on endosomes. COP I & COP II coat on golgi and ER membrane. Coat recruitment GTPase are a family of monomeric GTPase.

They include the Arf-protein, which is responsible for both COP I coat assembly and clathrin coat assembly at golgi membrane .

3.3.1.     SAR-I protein, which is responsible for COP-II coat assembly at the ER membrane.

Rab protein plays a central role in specificity of vesicular transport. They direct the vesicles to specific targets and then SNARE proteins to mediate the fusion of lipid bilayer. Rab proteins binds to other proteins called Rab effector which facilitate vesicles transport and membrane fusion. The SNARE proteins catalyze the membrane fusion reaction in vesicular transport . Vesicular fusion is mediated by interaction between specific transmembrane protein i.e. SNARE protein on vesicle and target membrane V-SNARE & T-SNARE respectively.

There are atleast 35 different SNARE’s in an animal cell .Each associated with a particular organelle in the biosynthetic, secretory or endocytic pathway.

SNAREs have been best charaterrised by in neurons, where they mediate the docking and fusion of synaptic vesicles at the terminal of nerve PM in the process of neurotransmission.

3.4.         Lysosome targeting:

Discovered and named by Belgian biologist Christian de Duve, who received the Noble prize in physiology medicine in 1974. Lysosomes are membrane-enclosed, spherical bodies, or vacuoles which is filled with different hydrolytic enzymes, including proteases, lipases, phosphates, glycosides, phospholipases and sulfates. All are called as hydrolases, because all enzymes are active at optimal pH i.e. 5.0 acidic environment. In order to maintain the acidic pH, the lysosome have H+ pump on its membrane, use's ATP hydrolysis as a source of energy to pump H+ ions into the lysomome lumen from the cytoplasm.

Lysosomes avoid self digestion by glycosylation of inner membrane proteins, which prevent their degradation. Lysosomes help in eukaryotic cells obtain nourishment from macromolecular nutrients. The lysosome are formed vesicles contaning hydroloytic enzymes and proton pumps bud off from the Golgi complex. Lysosomes allow interacellular digestion in eukaryotes and digested material crosses the lysosomes membrane into the cytoplasm.

The enzymes that are within the lysosome are made into rough endoplasmic reticulum, which are then delivered to the golgi apparatus via transport vesicles. Where they modified glycosylation lysosomal end are tagged for lysosomes by the addition of cromose label. When lysosome reaches the cytoplasm, fusion forms a secondary lysosome. A shortage of any lysosomal enzyme lead to lysosome diseases such as Tay-Sachs disease & Pompe's disease etc.

There are two types of lysosomes-Primary lysosomes-that do not contain any foreign particles or cell's content or membrane for digestion and secondary lysosomes are that which contain foreign particles or membrane for being digested.

Autophagy (Autophagocytosis) :

Greek word "auto" or ourself & "phagy" to eat. Autophagy is a process of self-eating and self degradation. The degradation takes part when the cell content & organelles is consumed by lysosomes. The cell produces vesicles called autophagosomes that captures and deliver cytoplasmic material to lysosomes. Autophagosomes are formed when the ER structures or cell content, are joining with lysosome and destroy the content captured and delivers the broken down material to the cytoplasm. Overall, autophagocytosis preserves the health of cells & tissues by degrading unused or damaged cellular content to new ones.

Endocytosis

When cell engulf large nutritional molecules form endosome and through which lysosome then fuse, where by it transfer it enzymes to break molecules. the broken down molecules ore then delivered to the cytoplasm by protein for later consumption.

Phagocytosis

When phagosome containing bacteria or any foreign object are fuse with lysosome. These objects are break down by lysosomal enzymes. Phagosomes are produced when the cell membrane of a cell surrounds a disease causing bacterium. Toxic or unused content in outside the cell.

3.4.1.     Protein Targeting to lysosome

The processing of N-linked oligosaccharide of lysosomal protein differ from that of secretory end PM proteins.A protein is destined for incorporation to lysosome or modified by mannose phophorylation and initial removal of mannose residue does not occur. The NAG –P are added to specific mannose residue M-P-NAG,while the protein is still in CGN.This is followed by the removal of NAG group leaving Mannose-6-phosphate residue are recognized by a M-6-P receptor on the TGN which directly transport of these proteins to endosome/to lysosome.The recognition determinants that leads to M-P depends on the 3-D confirmation of folded proteins and such determinants are called signal determinants or signal patches.

3.5.         Post translation transport :

3.5.1.     Mitochondria :

The word micochondria comes from the Greek mitos-i.e "thread" and condrion, i.e. "granule" or grain like. The word mitochondria coined by C.Benda. The mitochondrion (plural mitochondria) is a double membrane-bound organelle found in all Eucaryotic organisms including all animal cells and plant cells. The number of mitochondria in each cell varies enormously from just one mitochondria upto 10,000 mitochondria in some specialized types of cells.

Mitochondria have an ellipsoid i.e "oval-shaped" or "rounded rod-like" shaped. Mitochondria are continually active. They move and can change their exact shape. Mitochondria are double membrane bound structure between 0.7503mm in diameter membrane defines the external shape of the mitochondria called outer membrane of mitochondria (OMM) is permeable to oxygen, pyruvate, ATP & other molecules. Its thickness about approx 40°. The outer membrane is a relatively simple phospholipid bilayer, contaning protein structure called porins, which provide permeable to molecules of about 10 kda.

The inner mitochondrial membrane (IMM) has many flat or tubular folds or invagination called cristae. This inner membrane that form cristae are covered with many tiny "stalked particles" called inner membrane spheres whose head i.e the "Sphere" or "knobs" or part, is on the matrix side of the inner membrane. They contain a protein called F1, which written as "F1" attached on FO portion.

Many of the chemical reaction that take place within mitochondria occur on the inner membrane. It contains the electron transport  system (generate a proton gradient) and ATPase complex produce cellular energy in form of the ATP hence called Power House of the cell hence the inner mitochondrial membrane is the site of oxidative phosphorylation. The thickness of inner membrane layer is approx 40°. The volume between the inner & outer membrane is called the intermembrane space, or peri mitochondrial space. It has a high proton (H+) concentration, due to electron transport system of inner mitochondrial membrane. The space between the inner & outer mitochondrial membrane is approx 70°.

Matrix

The volume enclosed by the inner membrane is called "matrix". The matrix of each mitochondrion contains :-

Enzymes - of TCA cycle (tricarboxylic acid cycle)/citric acid cycle/the Krebs cycle & the Szent-Gyorgyi-Krep cycle. The only enz, involved in the TCA cycle that is not free in the mitochondrial matrix is Succinate dehydrogenase, which is located on the inner surface of the inner mitochondrial membrane.

Ribosomes - Ribosomes are of 70S types, as found in Procaryotic cells (bacteria) as opposed to the 80S type present in many plant and animal cells. They can synthesize proteins.

Matrix granules and mitochondrial mitochondrial DNA and many proteins & molecules  - Mitochondria have their own ds circular genetic material DNA and the facility to produce their own ribonucleic acids (RNAs) of ribosome & proteins and 22 tRNA, mRNA. All of the  mitochondrial DNA is maternal. Mitochondrial carries genes necessary for the synthesis of many, but not all, mitochondrial proteins.

Origin and Evolution

Mitochondria are semi-autonomous organelle (self replicating) and divide by binary fission similar as bacteria do. It is believed that mitochondria evolved from bacteria due to having many similarities with Bacteria (Procaryotic Characters. evidence )

  1. Membranes : Mitochondria have their own cell membrane just like a Procaryotic cell does.
  2. DNA : Each mitochondria has its own circular DNA genome, like a bacteria's genome, but much smaller. This DNA is passed from a mitochondrian to its offspring & is separate from the "host" cells genome in the nucleus.
  3. Reproduction :  Mitochondria divide by binary fission, the same process used by bacteria. Every new mitochondrion must be produced from a parent mitochondrion in this way, if a cell's mitochondria are removed, it can't build new ones from scratch. Proposed by Lunn Margulis, He suggested fission to endosymbiotic hypothesis in which mitochondria were originally prokaryotic (bacteria) cells, capable of implementing oxidative mechanisms that were not possible for eukaryotic cells : they endosymbionts inside the eukaryote in living form. The ability of these bacteria to conduct respiration in host cells that had relied on glycolysis and fermentation would have provided a considerable evolutionary advantage. Mitochondria (in mammals cell) may replicate their DNA and divide mainly in response to the energy needs of the cell, by binary fission. When the energy need of cell are high, mitochondria grows and divide and when energy use is low they are distroyed or become inactive. An individual's mitochondrial genes are not inherited by the same mechanism as nuclear genes. Typically the mitochondria are inherited from one parent only in higher eukaryotes, usually mitochondrial DNA come from the egg only i.e. from maternal a pattern known as maternal inheritance. In lower eukaryotes such as yeasts, both parents contribute equal around of mitochondria to the zygote thus in yeast mitochondria inheritance is biparental type.

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:

  1. ATP hydrolysis by Hsc70 chaperone proteins in both cytosol and the mitochondrial matrix is required for import of mitochondrial proteins (invivo only)
  2. 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).
  3. 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

  1. 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).
  2. 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).
  3. 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.

3.6.         Chloroplast

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.

  1. The outer chloroplast membrane.
  2. The inner chloroplast membrane.
  3. 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.

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.

Grana

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.

Key point

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.

3.8.         Nucleus

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.

Nuclear Import

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.

Nuclear Export

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.

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