Cellular Protein Traffic:

Transport of Proteins into Organelles:

Almost all organelle proteins are synthesized free from ER. The mRNA-ribosomal machinery that is involved in translation may be held in cytoplasm by Actin filaments or microtrabacular elements! They may be localized as in the developing oocyte of Drosophila and other systems.

· Mitochondrial genome has the ability to produce 13 polypeptide chains and also contain few URFs (unidentified reading frames). In addition, the genome contains genes for all tRNAs and rRNAs for mitochondrial ribosomes. Chloroplasts, besides coding for ribosomal RNAs and all its tRNAs, have the ability to synthesize at least 120 or more polypeptides. All of them are the residents of chloroplasts.

· The rest of the proteins, hundreds or more, required for mitochondria, chloroplasts, nucleus and Glyoxysome or peroxisomes have to come from cytosol, where nuclear coded transcripts meant for cell organelles are translated. These proteins have to be identified, sorted and delivered to their respective destinations post translationally.

· Each of these organelles is bound by outer and inner membranes (except peroxisomes which are covered by one unit membrane), which are structurally and functionally differentiated. Majority or all organelle proteins contain 12 to 70 amino acid signal sequences one at their N-terminal and at the other at adjacent regions. They can use two signal sequences one for the transport into the matrix and the other to transport into periplasmic or inner membranes.

· The organelles contain receptor cum protein channels where the outer and inner membranes are held to each other. These regions act as conduits for the exchange of molecules of different sizes to and fro. Organelle proteins synthesized in cytosol are recognized by their sequences organelle specific receptor protein and bind to them. Then they are transported to their respective destinations.

· The receptors located in membrane recognize the signal sequences of the protein, and bind to such sequences in a manner they are folded into a pattern that facilitates threading them into the channel. In this process chaperones help the proteins in threading the protein into organelles.

· While the protein is threaded through, it undergoes unfolding and as and when it enters into the organelle, it again reforms into its native conformation. In this process organelle HsPs subunits play important role in protein folding inside the mitochondria.

· The signal sequence of organelle proteins is different from those of proteins destined to be secreted or destined to be the resident proteins of ER and other membranes. There is little homology between them. Once they are threaded through the channel, the signal sequences are cleaved by signal peptidases located at the inner side of organelle membranes.

· Moreover, the signal sequences between different organelles are different and there is little homology between them. However the leader sequences contain a stretch of uncharged amino acids interrupted by basic amino acids, but lack acidic amino acids. The leader region my conformationally from a partial helical structure.

· If a cytosolic protein like di-hydrofolate (DHFR) reductase gene is reconstructed with N-terminal leader sequences of mitochondria or chloroplast and expressed in eukaryotic cells like HeLa cell or any plant cell, the DHFR protein ends up either in chloroplast or mitochondria, depending upon the cell type. This experiment clearly illustrates the importance of signal sequences in identifying the protein and delivering the protein to their respective targets.

Transport into Mitochondria:

Mitochondria energy transducing organelles of all eukaryotic cells. They are bound by an outer and inner membrane. The two membranes are joined to one another at regions of receptor cum channel proteins called attachment sites (old terminology). It is through such protein complexes mitochondrial proteins are recognized and transported into mitochondria.

Mitochondria by itself consist of three compartments. The central matrix, inner membrane with cristae and peri-mitochondrial space. Each of them contains different sets of proteins.

Mitochondria perse has its own genome. The mitochondrial genome size varies from organism to another, but the number of genomic copies ranges from twenty to fifty or more. But most of the mitochondrial DNA codes for 13 proteins that gets integrated into inner membranes as protein complexes. The genome also has few unidentified reading frames called URFs. It also codes for mit-ribosomal RNAs and 22 or more tRNAs. It has its own translational machinery. Yet for its structural and functional organization it requires more than another150 proteins, which are coded by nuclear genome, translated in the cytosol and transported into the organelle.

Mitochondrial proteins coded for by the nuclear genome posses signature sequences. Some destined into matrix have one N-terminal sequence and those destined to inner membrane or peri mitochondrial space contain second signal sequences, one for the entry into matrix and the other directed to inner membrane or to perispace. Perhaps, mitochondria from all organisms from primitive to the most advanced ones may contain the same but a specific signal sequences for each and every mitochondrial proteins.

Mitochondria membranes at attachment point consist of a channel protein complex; one complex is Transporter of outer membrane called TOM and another Transporter at inner membrane called TIM. TOM consists of more than 9 subunits of more than 500KD. The thickness of the channel is 188 Å and the central channel is about 20 Å. Tim is a multi protein complex consists of channel made up of 17 and 23. The TOM 40 and TIM are in direct line with one another but they don’t interact with one another. Tom is associated with many protein subunits such as tom-5, 6, 7 (assembly factors) and also with Tom 37, 71 and 70 (one type of receptors) and Tom 22 and 22 and 22 (another type of receptor). Tim is also associated with several proteins such as Tim 17, 23, 44, 22-54, 9, 10 and 12), involved in transport of protein delivered by Tom 40.

To begin with as polypeptide chains emerge out of the tunnel in large ribosomal subunit, the free end can assume any conformation, but before the ends assume any structural form they are bound by HsP proteins to and prevent such folding. At the same time a specific receptor binds to signal sequence and directs the protein to mitochondrial transport receptor located at the outer region of the channel. During transfer receptors recognize specific mitochondrial signal sequences in the protein and binds to them, then they are threaded into the channel by ATP dependent manner. Then the N-terminal region of the protein is handed over to intermediate Tims (Tim 9,10 and 12 located in interspace or what is called peri-mitochondrial space), then the protein is transferred to Tim 22-54 channel proteins and the protein is transferred to mitochondrial matrix. But the entry is facilitated by the HsP70 and MGE (= Grp-E), facilitated by Tim 44, which not only binds to the N-terminal part of the protein but also pulls the protein inwards. As the N-terminal segment of the protein enters the matrix, the signal sequences are cut. If the protein destined to go into inner membrane the protein uses the second signal sequence.

The protein subunit IV of Cyt.C1 has 61 amino acids at the N- terminal end. The first 32 amino acids are used to transport into mitochondrial matrix and the next 19 amino acids are used for targeting protein into inner membrane.

Cyt.C1: NH2-----32 aa-II-29aa-I—DHFR---------------------COO ^-

A recombinant DNA coding for DHFR containing two mitochondrial signal sequences, one for matrix and the second for peri-mitochondrial space in to a system, the DHFR ultimately lands up in peri-mitochondrial space.

Some mitochondrial proteins have two signal sequences, one at N-terminal end, which facilitates the entry into the mitochondrial matrix. The matrix signal sequences are cleaved. The second sequence facilitates the entry into peri-mitochondrial space, there water soluble protease cleaves the second sequence, but it requires Mg ^2+. Transport to each of the destinations requires ATP energy. An electropetential gradient across the membrane is also required for the protein to be transported across the membranes.

Cyt.C oxidase subunit IV- leader sequence:

3HN-M - -+- + - +32 - - - - - - - -A-49-IS-T - - - - - -A-61-I- - - -

----- Charged--- ---- uncharged---

Cyt.C oxidase: NH2-MLSLRFFK PATRTLCSSRYLL//

Cyt. C1: MFSNLSKRWAQRTLSKSFYSTATGAASL-

SGKLT//EKLVTAGVAAAGITASTLLYADSLTAEA//

Transport into Plastids:

Plasmid perse develop from proplastids and require many signals for the development. Among the signals light is very important.

In plants, nuclear coded proteins, for both mitochondrial and plastids are synthesized in the same cytoplasmic space, using specific signal sequences and specific receptors, they are differentially sorted out and targeted to their respective destinations. Once they enter the inner space of the organelles they are further targeted to their respective inner membranes, some times few specific proteins move into periplasmic space.

Plastids exist in different forms such as proplastids that develop into fully formed plastids, either as chloroplasts, chromoplasts, elaioplasts, leucoplasts or proteinoplast. A green plastid, the chloroplast is highly differentiated into stromal lamellae and granal lamellae. These are embedded in a fluid called stromal fluid. Chloroplast posses it own genome, whose size varies fromone system to the other, however though the size is limited, they contain several copies of the genome. The plastid genome encoded with 2rRNA genes, 22 or more tRNA genes and 120 or more chloroplast resident genes; many of them are organized into thylakoid membranes for light harvesting. The rest of the proteins may be in hundreds, they have to come from nuclear coded and translated in cytoplasm

These proteins as in mitochondrial proteins, as they emerge from the surface of large subunit of robosomes, they are bound by HsPs to prevent unwanted folding. At the same time receptor proteins to specific signal sequences bind them. Then they are handed over to receptor cum translocator channels found at eh junction of outer and inner membranes. Such receptor-channel protein complexes are found in large numbers to facilitate the transport of proteins. Matrix proteins require just one signal, but those proteins that are destined to thylakoid membranes, contain two such sequences, one for the matrix and another to thylakoid membranes similar to mitochondria.

Peroxisomal Proteins:

Peroxisomes are vesicular structures containing proteins called Peroxins. It has been estimated that there 26 genes that are involved in the development of peroxisomes. Their main function is detoxifying peroxides into water and oxygen by catalases. But also they peroxidase enzymes that can generate hydrogen peroxides, but they are removed immediately by catalases.

All organelles, which have membranes as their structural component, require specific pre-existing particles for their growth and multiplication. Pre-existing peroxisomal membrane vesicular structures are required for the assembly and growth of peroxisomes. Peroxisomes or Glyoxysome are bound by single unit membranes similar to that of Lysosomes.

Those proteins, which are synthesized free from ER, which contain SKL sequences at C-terminal ends, are targeted to peroxisomes. The SKL sequence is specific for peroxisomal proteins, but now it is known SKL has nothing to do with it. The membranes have receptors that recognize SKL sequences at the C-end of the protein, now it is called PTS-1. Now it known that the SKL is not the sequence that is responsible for loading proteins into organelle. Instead it has another nine amino acid sequence at N-end of the protein called PTS2. There can another sequence called PTS-3. The membrane contains channel proteins called pex14p and pex13p. The receptor proteins pex5p and pex7p recognize proteins that have such sequences and bind to them then the proteins are translocated across the channel into the organelle in fully folded form. The PEX5p binds to the peroxisomal protein and delivers it across the channel. After the delivery it returns to cytosol.

Transport into The Nucleus:

Presence and absence of the nucleus makes organisms distinguishable as prokaryotes and eukaryotes, one without the nucleus and the other with the nucleus respectively. Nucleus is a vital cell organelle and contains genomes with all the genetic information. Nuclear structure is highly organized and executes all cellular functions through genetic regulation of host of genes by the way of transcriptional activation or repression and processing of transcripts and transport of them. A single mutation can render the nuclear function to damaging results. Nucleus, with chromosomes, nucleolus and nuclear sap, consists of hundreds of proteins, all have to be imported. Many molecules of various sizes are transported into the nucleus; similarly various molecules are transported out into the cytoplasm.

Structurally the vital contents of the nucleus are enveloped by a membrane system that is very complex with highly organized structures. It is the membrane acts as the gateway for the movement of proteins into the nucleus and out of the nucleus. The structure in the membrane that acts as a portal for such movements is pore complex.

An artistic view of Nuclear Pore Complex

This picture shows the components of the pore complex in –a longititudinal sectional view

The picture is a sectional view with components labeled

The nuclear pore complex is a huge structure (120nm in diameter) with hundreds of proteins; the molecular weight of the total mass is 125 million Daltons or more. The pore complex is constructed like an annular concentric rings looks like a wine barrel with an upper ring and lower ring of eight subunits each. The upper top ring faces the cytoplasm and lower or bottom ring faces the nuclear sap. Protein filaments emanate from the upper ring, so also from the bottom ring, but the filaments from the bottom ring are connected to each other in the form of a ring. It contains a central channel of 40 nm size runs all along the length of the barrel. The central channel is linked to outer coaxial ring of proteins by eight spokes. The coaxial ring in turn is connected to nuclear membranes by radial arms. The space between the radial spokes also provides free space for the movement of smaller molecules. The central pore complex has cytoplasmic Iris and nucleoplasmic Iris connected to cytoplasmic ring and nucleoplasmic ring respectively.

A network of structural proteins called Lamins supports the nuclear membrane at its inner surface. The number of pore complexes can expand and contract according to the needs. Average number of pore complexes per nucleus is ~3000.

The pore complex is responsible for the import of several kinds of nuclear proteins and export of several species of RNAs, ribosomal subunits and many snRNPs. The number of proteins imported per pore complex per minute is remarkable. One hundred Histones are imported per minute per pore complex. Non-histones 200, and riboproteins 150 per minute imported. The same pore complexes export ~5 ribosomes per minute per pore complex, mRNAs one per minute. The database estimates 6000 to 10000 different proteins are imported from cytoplasm into the nucleus.

Molecules of 5 to 500 Daltons size easily move across the pore complex, thus nucleotides, ions many other molecules have easy access into the nucleus. But proteins of more than 50KD (size of ~5nm) cannot diffuse passively, but they are transported in ATP dependent manner (Active process). The central channel is exclusively used for the transport larger cargo by expansion and contraction mode. They can transport gold particles of size~ 20nm through central core. The pore complex transports ribosome of 120 x 200Å size. The central channel has a cytoplasmic iris connecting cytoplasm and a nucleoplasmic iris connecting nucleoplasm. Though the pore complex allows components passive diffusion, it is always facilitated. Active transport is always a facilated transport. Cytoplasm contains 20-30 thousand different proteins, but only those proteins that are required are transported and not others. So also export is directed and facilitated.

Import of Nuclear proteins:

Transport of proteins into the nucleus is determined by specific signal sequences called Nuclear Localization Sequences (NLS). It appears most of the nuclear proteins have a common signature sequences for the import. Similarly those proteins or RNA species destined for export should have similar signature sequences called Nuclear Export Sequences (NES). Proteins involved in import are called Importins and those used in export are considered as Exportins. Here are some of the signal sequences of proteins destined to the nucleus. Export protein sequences have a conserved 10 amino acids with conserved leucines.

SV40 T-antigen: PKKKRKV.

Polyoma T-antigen: PKKARED.

Nucleoplasmin: KR-10 aa-KKKK.

SV40-P1: APTKRKGS.

Import of nuclear protein requires a host of proteins called Importins. But the importin-α, β, and β3 are important. The importins are located at the orifice of the pore complex. Importin-α binds to NLS sequences then transfers to importin-β, which in turn delivers to nucleoporins (Nuc-Ps) found at the nuclear membrane in the pore complex. They are involved in inward movement of cargo. There are a many nucleoporins involved in translocation of proteins into the nucleus, it requires the input of ATP energy. There is another group of importins called importin-β3, another group is called Transportins. which not only binds to NLS sequences but also delivers the cargo to nucleoporins. Transportin M9 is mainly responsible for the transport of hnRNPs. Importinβ3 is belied to responsible for the import of riboproteins. There are more importins, which carry signal molecules into the nucleus for activating transcription. Nucleoporins have repeated motifs such as GKFG, FG, and FxFG. These motifs help in binding to importin-β and driving the cargo inside.

Another cytosolic factor that supports translocation is Ran. It is a GTPase protein. Ran is a monomeric G-protein. The Ran-GTP complex is found associated with importins at cytosolic side and also found at the nucleoplasmic side. The Ran-GTP is active, when hydrolysed it provides energy for the movement. The Ran-GDP, is inactive, it will be activated only when GDP is exchanged with another GTP by another factor called GEF (GTP exchange factor).

Export of Nuclear Cargo:

Similar to importins there are Exportins molecules that are specific each type and they are responsible for the export of mRNAs, ScRNAs, SnRNAs tRNAs, many proteins and ribosomes into cytoplasm. Export of protein requires signal sequences such as NES, a ten amino acid residues stretch that is rich in Leucines. Export of components is species specific and requires specific nucleoporins aided by Ran-GTP factors found at nucleoplasmic surface. Specific

factors determine which RNA has to exported, ex. HIV mRNA intact transcript (unspliced requires Rev proteins which bind to sequences called Rev response elements (RREs). Similarly most of the mRNA, which are spliced, requires cap proteins bound to the Guanine nucleotide found at 5’end for transport of processed mRNAs. TRNAs transport again requires specific factors for transport. It is interesting to know some proteins such as poly-A binding proteins; PAB-II and RNAs (especially snRNAs) that are transported out of the nucleus are transported back. Such transport requires specific importins and exportins, which act to and fro transport.

Transport of Proteins Via Membrane Traffic:

Whether proteins synthesized on ER or in free state in cellular milieu, localized or not, each of the proteins have distinct destination and they have to be directed and targeted specifically and correctly. A single cell can easily produce 40,000 or more number of proteins. If alternate splicing of mRNAs, splicing of intein containing proteins and processing of poly-proteins, the total number can be anywhere 60,000 or so. Cells have endowed with facility and innate ability to sort out each and every protein and transfer to their specific destination. Though the process is complex and many of the events are yet to be understood, the overall or generalized mechanism is emerging from studies.

· Proteins, that are synthesized and threaded through ER, have several destinations like ER itself, Golgi, lysosomes, vacuoles, plasma membranes and secretion to extracelluler space. The transport, from ER to Cis-Golgi to median-Golgi to Trans-Golgi, to transport vescicles and then to plasma membrane, forms the default pathway.

Proteins that are synthesized in free state localized or otherwise are sorted and transferred to their specific destinations like the Nucleus,

The diagram is an excellent EM of Golgi membranes associated with ER

Mitochondria, Chloroplasts (in plant cells), and Glyoxysome or Peroxisomes.

Cell organelles like chloroplasts and mitochondria themselves synthesize their own proteins within the organelles, and they have to find their specific destination to organize into specific structures such as thylakoids in chloroplasts, cristae in mitochondria and soluble fluid within the organelle.

The picture

The picture depicts the relationship between the ER and Golgi

If one visualizes the cellular microcosm and its components, which are produced and degraded every minute of its existence, it is as amazing and bewildering as the macrocosm of the universe.

· Proteins that are synthesized in free form, i.e. not on ER, are targeted to different organelles and transported to their respective destinations. But proteins that are synthesized on ER have various targets. First they have to be folded properly, if needed they are processed and modified in the sense they are added with carbohydrate, acetylated, or phosphorylated and localized in the membrane in specific orientation or they may be just placed in the lumen. Gradually as the more and more of proteins are transported into the ER lumen, with modification the membrane start extending into ER free from ribosomes, called SER. It is during these transitions RER to SER the proteins are marked, modified or being in the process of modification and marking. Endoplasmic membrane network is supported by microtubular skeletal network.

At a docking site of ER, the binding of one SRP-mRNA-ribosome complex, it is fairly assumed that only one kind of proteins is synthesized and threaded into the ER lumen. Once such cluster of proteins build up they are modified and bunched together; then the ER membrane expands into a SER surface, then it is pulled towards cytoplasm into a sac like structure called a vesicle. Many such vesicles from SER at various sites develop and pinched off. Vesiculation from SER is performed by a set of proteins called COPs or coat proteins of specifc kind. Such vesicles are then fuse with one another or fuse with proximal region of Golgi complex, which is called cis surface.

Default pathway

This is another default pathway

· Golgi complex is a bunch of membranous sacs called cisternae, like saucers, stacked one above the other. The Golgi membranes near to SER are called cis Golgi and the membranes found at other extreme end are called Trans Golgi. In between cis and trans Golgi membranes many layers of sacs are found called median Golgi. The Golgi membranes near the nucleus curved away from it, but membranes facing SER are curved towards them. Each of the Golgi membranes can be identified by marker enzyme found in them. The cis Golgi sacs contain Glycosylamine acetyl transferase and mannosidase. Medial sacs contain N-acetyl glucosaminidase, mannosidase-II. Trans and trans Golgi network contain galactosyl transferase, mannose6-phosphate receptors, sialyl transferase and acid phosphotase.

These two diagrams depict endocytosis of living materials and nonliving objects.

As in the case of ER, which is supported by microtubules for their stability, the centrosome complex and its elements have a role in the stability of Golgi apparatus. Golgi bodies are always in close proximity to centrioles. Use of colchicines and similar drugs to destabilize the microtubular assembly also disturbs the assembly of Golgi membrane. As in the case of ER, Golgi membranes also disperse into membrane vesicles during mitosis or meiosis. Are these dispersed membrane vesicles do have some markers so as to reassociate to form the fully developed systems?!

This picture shows the mechanism of endocytosis of LDL receptors and once they are delivered into cytoplasm they are returned to the membrane surface.

This is an excellent EM showing the cytoskeleton fibers involved in constriction and transport of materials

This diagram is a simplistic representation of reticular endothelium spread all over but supported by microtubules; Golgi apparatus is just sitting very adjacent to the nucleus; even the Golgi membranes are supported by cytoskeletal net work.

This EM shows the fluorescent labeled cellular membranes including Golgi

· Once the cargo containing vescicles from SER fuse with cis Golgi, proteins go though further processing’s. Step by step proteins in median Golgi membranes are subjected designed modifications, they are also sorted out and collected at lateral regions of the membranes and they look like swollen structure. From the top Golgi membranes the proteins are again packed into vescicles and budded out. Again this vesciculation is performed by a set of coat proteins. As the processing is performed in stepwise manner the assorted proteins are packed into vesicle, mostly from the later side and budded off. Thus a series of vescicles are transferred from the Cis Golgi to the median Golgi one after another and finally to the trans Golgi. The stacked membrane sacs are interconnected and fluid in them exchange. By the time protein reach trans surface they are already marked and they loaded into vescicles, which are released from the trans surface. This vesiculation from SER to Cis Golgi to median Golgi and to trans Golgi is performed by a set of coat proteins. These vesicles pinch off from the anterior membranes and handover them to the next membrane sac, then another set of vesicle develop from the cis Golgi membranes and they fuse with the next underlying Golgi membrane, thus the proteins are step wisely modified and processed and transported to the trans surface like a bucket brigade to be released ultimately to their specific destinations at the distal surface of the Golgi. This type of movement from SER to Cis, from cis to median and from median to trans is called forward or anterograde movement. But membranes from Golgi also move towards ER by a series vescicles. This movement is called retrograde movement. Again this process is performed by another set of coat proteins or cops.

The vescicles produced at trans surface are loaded with proteins that are destined to specific location of the cell. The proteins that pass though various stages of processing and various levels of Golgi are ultimately loaded into vescicles. Some of the proteins that are transported from one level to the other are marked. Example lysosomal proteins are phosphorylated at specific mannose residues, so phosphorylation of mannose is marker for lysosomal enzymes. ER and Golgi membranes contain transmembrane receptors for mannose phosphate residues, facing the lumen. Thus the lysosomal proteins are held onto receptors. Specific Cops bud off such clusters in the lumen again. Similarly other proteins are also sorted and loaded onto membranes, which are pulled away from the membranes in the form of vescicles, which are again coated with Cops.

· Vesicles carrying lysosomes are transferred to early endosomes or late endosome to develop into lysosomes. Vesicles loaded with lysosomal enzymes are marked with specific receptor proteins, so their destination is already determined. Similarly the proteins for plasma membranes are carried by coated vescicles and transported to plasma membrane surface, where the vesicles fuse with plasma membrane. Specific proteins again aid the fusion. Similarly those proteins that are destined to secrete out of cell surface are transported as coated vesicles and they fuse with plasma membrane in such a way all the contents of vesicles are unloaded into exoplasmic region. Some vesicles carrying certain cargo like acetylcholine or such compounds are transferred to synaptonemal membranes. Some vesicles store certain components where they are stored as granules in storage vesicles. Whatever vescicles generate trans Golgi membrane is coated with specific coat proteins, which have marked destination. The most default path way is from ER to Golgi, and from Golgi to plasma membrane.

Cell surfaces contain receptors, which have external domains with sites for the binding of specific ligands. Each cell depending upon the cell type posses thousands of different receptors. Pit areas mark the cytoplsmic side of the receptors. When the ligand binds, the membrane pulled inwards carrying the ligand inwards as endosomes, by a process called endocytosis. Some times cell surface bound components are engulfed as phagocytosomes. Another way of membrane invagination engulfing liquid from out side is Pinocytosis. The vesiculation and inward movement of vesicles is also controlled by a set of proteins. From plasma membrane to lysosomes, from endothelial reticulum to Golgi complex, from Golgi complex to lysosomes, plasma membranes and secretion to extra cellular space is a designed traffic flow of membranes carrying proteins from one destination to the other delivering different proteins to different sites. In this process membranes are budded off, membranes fuse with one another and in some cases membranes disperse into small vesicles only to be reformed at certain stages.

· The flow of proteins from the site of synthesis to their destination is follows two distinct pathways. One that the proteins synthesized in free state are directed to theirs respective destination such as chloroplasts, mitochondria, peroxisome and the nucleus, is more or less guided by HsPs and receptor proteins. The other pathway is that though the protein synthesis starts in free state, the N-terminal sequences of them determine, their further fate, they are placed on ER and the synthesis continues and all those proteins synthesized on ER are threaded into ER lumen. The number of proteins synthesized on ER is substantial and their destination is also varied; their destination includes all the net work of endothelial membranes, Golgi bodies, lysosomes, plasma membranes, exoplasmic space, probably all other organelle membranes proteins and all those transport membrane vesicles that transport that transport proteins. Most of the proteins that are synthesized on ER and threaded into ER are transported through vescicles. Not with standing, a large number of components from exoplasmic space find their way into cells, by inward flow in the form of membrane bound vesicles and the same are distributed to their destinations.

Not all thousands of proteins that enter from ribosomes into ER, from ER to Golgi are transported out of them. Many, reasonable all the proteins required for structural organization of membranes, proteins required for protein modifications and marking other proteins for transport out of Golgi, remain in ER and Golgi bodies, they are called resident proteins. These proteins have transcellular domains, so their hydrophobic regions are anchored into the membranes. One end of the protein, a major part of the protein, is towards the lumen and its domain is towards cytoplasm. Many of such proteins are receptors and they are responsible for binding to specific lumen proteins, they move along with the flow of membranes from one region to the other region, till they are released from their cargo. The cytoplasmic

domain has certain sequence that acts a signature for the movement of resident proteins from ER to Golgi and back to ER. Specific carrier or transporter transfers all the inputs required for modifications of lumen proteins from cytoplasm into lumen.

· A set of related proteins are involved in binding to cytoplasmic surface of membranes and pull the membrane into a bud or vesicle carrying fluid and proteins. This happens all the time at plasma membrane, at ER, at Golgi membranes and endosomes. It is the vesicles carrying proteins are budded off from the said membrane surfaces, they are also transported to target sites and they fuse with their target membranes. This kind of membrane flow carrying protein as cargo takes place in the cell all the time and each of these events take place at their own pace.

The vesicle budding off at plasm membrane surface and vesicles forming at Trans-Golgi are mediated by clathrin-coated structures. Preformed clathrin coated pit areas determine their site. Another mode of vesicular traffic takes place at the surface of SER and Golgi. Once the SER membranes are loaded with proteins they are loaded at one side of the membrane and the same budded off carrying a group of proteins in the form of transition vesicles. Budding at SER surface is due to certain protein assembly called coatamers COP-II. These vesicles fuse with cis-Golgi membranes at which proteins are subjected to modification in terms of removal certain sugars and addition of certain sugars. Addition of phosphate groups to certain sites. Then the Cis-Golgi bud off vesicles, which fuse with median Golgi membranes, where more modification takes place. Proteins with O-linked glycosylation take place only in Golgi cisternae not anywhere. From Cis-Golgi proteins are clustered into similar kinds or same types and transferred to Trans-Golgi. It is here at Trans-Golgi final sorting of proteins is done according to their structural or functional features. Earlier the proteins are marked, as in the case of all lysosomal enzymes; they are phosphorylated at two mannose sites. Mannose-P is a marker, the resident receptor proteins found in the membranes facing luminal side bind to mannose phosphate group, thus all lysosomal proteins are bound and localized at a given region. Similarly other proteins are also localized. From the Trans-Golgi membranes proteins are further grouped and loaded into membrane vesicles and budded off to generate a large number of vesicles at Trans-Golgi surface called Trans-Golgi Net Work (TGNW).

The vesicles that develop from Cis-Golgi onwards to Trans-Golgi use another kind of coatamers called COP-I. COP-I performs forward movement, but also backward movement i.e. from trans-Golgi to Cis -Golgi to ER. But COP-II performs only forward movement is called Anterograde movement. Backward movement is called Retrograde movement.

Clathrin Coat proteins:

1. Clathrins: Large subunits 180KD and Small subunits (35KD). The large subunit, with small subunit, generates a network of triskelions linked to each other forming cage around the membrane with a hexagonal pattern. A triskelion consists of three light chains and three heavy chains organized into triradiant structure that form network of polyhedral coat protein as a coat anchoring the vesicle. Clathrin coated vesicles form at form at coated pits.

2. Adaptor proteins: AP-1, AP-2, AP-3 and AP-4. Each adaptor protein complexes are heterotetramers made up of adaptins.

Adaptins:

AP-1: γ1, β1, μ1A, σ1; associated with mannose marked vesicles.

AP-2: α, β2, μ2, σ2; associated with Clathrin coated vesicles at Plasma membranes.

AP-3: β3, δ, μ3, σ3; associated with signal transduced secretory vesicles.

AP4: ε, β4, μ4, σ4; associated with clathrin coats at Trans-Golgi membranes involved in transcytosis in polarized cells.

Among Adaptor proteins AP2 is the most abundant class of proteins, mostly found at plasma membranes. AP4 is associated with Trans-Golgi membranes. AP1 is generally associated with vesicles that transport lysosomal proteins with Mannose markers. AP3 associates with signal response secretor vesicles such as synaptic vesicles that form at endosomes. This type of APs is also found on secretory vesicles and those that transfer cargo from Golgi to lysosomes.

COP proteins or Coatamers:

COP-1: seven subunits form complex of ~700KD. The Beta Cop protein has homology to that of adaptin beta proteins.

COP-II: Sec 23p, Sec 24p (Tetramers 400KD), Sec13 and Sec 31(700KD), plus it is complexed with another protein called Sar 1p. The Sar 1P is a monomeric G-protein. Sec 23-P is GTPase activating protein (GAP). Sec 24 is recruiting protein.

The COP proteins exist in different forms having different functions.

Formation Clathrin Coated Vesicles:

Plasma membranes are studded with receptor proteins and many trans membrane proteins. Then specific receptor proteins are associated with specific adaptor protein, the adaptor proteins in turn are associated with clathrin skeletal proteins. They are the fore runners of Clathrin coated vesicles. Such regions are called coated pits. Such coated pits with different adaptor proteins are found as patches all along the inner surface of the plasma membrane. In clathrin coated pits β-adaptins bind to di-lycine (KK) sorting sequence of the receptor at the cytoplasmic face; at the same time they also bind to triskelions.

This diagram shows how a clathrin coated vesicle it formed and budded of into cytosol

This picture shows how a Golgi can be formed by budding from SER and fusion of the vesicles into cis membrane surface of the Golgi complex. Similarly traffic from one membrane of the Golgi to the other in forward and backward direction, then the matured and modified proteins are packed into vesicles at trans surface of the Golgi complex and budded off.

Adaptins μ2 in AP2 complex recognize tyrosine (YY) based sorting signal sequences. In this case phosphorylation of adaptins triggers internalization by clathrin-coated endocytosis. Similarly α & γ including δ and ε involved in vesicle formation by clathrin mediated coats.

Similarly AP1 complex is involved in producing a clathrin coated vesicles in carrying mannose-6 phosphorylated lysosomal proteins. But a variant of AP1 complex containing μ1B instead of u1A is found in polarized epithelial cells, where the vesicle carrying a cargo is carried from apical to basolateral surface. Again AP4 is associated with trans-Golgi membranes. However all these contain an additional protein called AP180; this protein decides the size of the vesicle. The above examples suggest that the adaptors are ultimate deciders, which cargo to be carried and to which destination.

COP-1 Coated Vesicle Formation:

COP-1 coated vesicles contain a seven protein (coatamer) complex. . The coatamers form two layers of proteins, the inner and the outer. One of the components of COP-1 is β-Cop. This protein has homology to clathrin coated adaptins β and β’-adaptins, which suggests that the β-cops play a similar role by contacting the signal sequences of the receptor proteins on one side and on the other side they contact outer cop coat proteins; the outer coatamers are analogous to clathrin coatamers. COP-1 coated vesicles perform both retrograde (mostly) and also anterograde (forward) movement.

COP-II Coated Vesicle Formation:

COP-II coatamers consists of sec23p and Sec24p as one complex of 700KD and the other complex sec13p and sec31p of 400KD. This also consists of Sar 1p. Sar1p is a GTP binding protein, Sec1p is GTPase activating protein (GAP), and it acts on Sar1p. But Sec1p is a protein responsible for recruiting the cargo proteins. COPII complex are responsible for the vesicle movement from SER to Cis-Golgi. Most of the COP coated vesicles have a size of 60-90nm.

How Vesicles Budd off and Fuse:

Proteins at different membranes surfaces at different sites (patch) get loaded and they are pulled into coated vesicles of different kinds, enclosing the cargo. Then they are transported (guided mode?) to their destinations, where they come in contact with another membrane surface either in the form of a specific vesicular membrane or a cell surface membrane; and fuse to deliver their cargo. There are a number of proteins involved in budding and fusion.

Budding Proteins and Budding:

ARF: ADP-Ribosylation factor for COP I and Clathrin coated vesicles.

Sar1p: A related factor for COP II coated vesicles, which perform function similar to ARF.

ARF is a GTP binding protein. It has binding site at N- terminal region. Its N-end can be myristoylated, provided the N-end opens out, so it can get inserted into a membrane. But opening of N-end depend upon the binding of GTP. When it bound by GTP the N-end of the ARF opens up and it inserts into the membrane. When the GTP hydrolyses to GDP, it becomes inactive and gets free from the membrane. Binding of ARF to membranes provide surface for the binding of coat proteins. Whether the COP-I or Clathrin associated proteins bind to membranes in association with ARFs, the membrane is deformed and pulled into a sac like structure, but the pinching off of the vesicle is performed by another protein called Dynamin. Dynamin is a contractile kind of protein. In GDP form it binds to coat proteins, but it is activated by GTP binding. Activation of Dynamin results in complete constriction and release of the cargo loaded vesicle.

Diagram shows components and events in membrane tethering

The figure shows various SNAP and Syntaxins involved in membrane transport

Transcytosis

A similar process operates with Sar1p associated with COP-II proteins. It is also GTP binding protein, but it does not need GTP binding for insertion of its N-terminal end by myristoylation. Activation of this protein by GTP leads to vesicle formation and Hydrolysis of GTP by Dynamin pinches off the vesicle from the membrane. ARFs in the released coated vesicles hydrolyze the GTP to GDP; this releases all the coat proteins from the vesicles. Some more proteins like Amphiphysin and Endophilin can also cause vesiculation but requires phosphotidyl-inositol for interaction in the membrane.

Vesicle Fusion and Fusion Proteins:

Once vesicles are released they are transported to their targets. Whether any actin microfilaments or microtubules are used for directed transport, though few years back was uncertain, now it is known many types of vesicles are transported using cytoskeletal elements.

One type of proteins are involved in recognition of specific membrane to membranes. Second type of proteins is involved bringing two membranes together. The third type of proteins is actually involved in fusion.

Rab proteins:

Rab is monomeric G-protein. Its C-end is prenylated and anchored in the membrane. Rab proteins are related to Ras proteins, which are kinases. There are as many as 30 Rab proteins. Rab gets activated when it is bound to GTP, but inactivated when bound to GDP. For activation it requires proteins like GAP and GEFs. These Rabs are specific-to-specific types of membranes. The following Rab proteins are involved indifferent transport vesicles (tentative). Rab proteins have their respective Rab effector proteins or docking proteins on target membranes, for proper effector function.

An overall view of intracellular transport involving Rab proteins

Rab 1 and 2: From ER to cis Golgi.

Rab 6: From cis Golgi to ER.

Rab 30, 33b, and 36: From cis to median to trans Golgi.

Rab 10, 12, and 13: From trans-Golgi to trans-Golgi network.

Rab 9: From trans Golgi network to Lysosomes.

Rab 4 and 5: exocytosis and endocytosis respectively.

Rab 7: From late endosome to lissome.

Rab 15, 18, 20 and 22: From early endosome to late endosome.

Rab 3 and 8: From TGNW to PM but regulated.

Rab 8 and 37: From TGNW to PM secretory (constitutive).

RB 11: From RE to exocytosis and from EE to TNG.

Tethering Complex: It is complex of proteins ranging from 5 to 8 subunits. There are different kinds of tethering complexes. Normally they are found very near to the membranes (i.e. target membranes). These proteins actually recognizing a specific Rab protein bring the vesicle membrane and the target membrane together. Rab protein provides the identity and facilitates tethering process.

20S complex: this complex is located on the target membranes. It consists of SNF protein that is sensitive to a Sulfhydryl agent called N-ethyl-maleimide. SNF is a soluble ATPase. It is homologue of yeast Sec18. This complex is associated with another protein called SNAP; it is so called because it is a Soluble NSF Attachment Protein. SNAP is anchored in the membrane. The receptor for SNAP is called SNARE.

The figure simplistic representation of V-SNARES from the donor membranes with coated vesicles and t-SNARES with target membranes involved in transport and docking.

Snare in different configurations

SNAREs: SNAREs are filamentous proteins associated with 20S complex of proteins. SNARE, which is localized (anchored) in the membrane. There are two types of SNAREs, one from the vesicle called v-SNARES and another is t-SNARES from target membranes, which are distinctly different from each of these membranes. The t-SNARE consists of Syntaxin 1A and SNAP25B (note that this SNAP25B is distinctly different from the SNAP i.e. soluble NSF attachment protein). Such SNAREs are also found in almost all systems. The vSNAREs are made up of synaptobrevin and SNAP 25B and they lie parallel to their membrane surface. VSNAREs and tSNAREs are different from one type of membrane to the other and their sub components vary from one another.

Fusion Process:

Once the Rab mediated docking takes place with the assistance of Tethering complex (each are specific to their transporter membranes),

The diagram depicts the docking of vesicle with V-SNARE with the membrane containing t-SNARE; after docking fusion proteins join to perform membranes fusion, where fusion proteins and V-SNAREs and t-SNARES are released.

An artist diagram of t and V SNARES docked on to a membrane

the SNARES from two membranes i.e. donor and target, coil to each other and pull the membranes close to each other in such a way the membranes bind to each other. This leads to the fusion of lipid layers and exchange of lipid components they join to generate a joint membrane. And release the cargo into the vesicle or to exterior space. This leads to the release of the SNAREs. Thus the proteins that are synthesized on ER are transferred into ER lumen and then they are modified at different levels and they do so while they are transported from SER to cis-Golgi.

This is an exotic 3-dimensional view of membrane transport to targets.

From the Cis-Golgi to median-Golgi to Trans-Golgi to Trans-Golgi network. The proteins are marked by specific modification and specific cargo carriers called receptors hold them on to the membrane in the lumen; they are loaded and sorted to each kind or many of the similar kind as one lot. This leads to the bundling of those sorted ones into a vesicle by specific coat proteins and they are budded off. On the other end cargo is carried from the plasma membrane and budded of into the cytosol. Such vesicles are transported to specific destinations where they are unloaded. Some of the vesicles that unload their cargo return to their old destinations. Example, lysosome receptors, secretory protein receptor that end up in plasma membranes return to their original locations. So also SER vesicles after transferring to cis Golgi or median Golgi return to their SER loci.

Protein carrying membrane transport in Plants; a brief but a lovely self-explanatory diagram cum review of a plant cell