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1. Details of Module and its Structure
Module Detail
Subject Name <BOTANY>
Paper Name <Cell Biology>
Module Name/Title < Endoplasmic reticulum: synthesis of secretory proteins, processing and transport by rough ER >
Module Id <6b>
Pre-requisites Basic knowledge about plant cells, their contents and basic idea about cell organelles
Objectives To make the students aware of the synthesis, processing and transport of proteins by rough ER.
Keywords Secretory proteins, protein targeting, COP II vesicles, protein folding
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2. 2. Development Team TABLE OF CONTENTS
1. Introduction
2. Targeting proteins to ER
3. Insertion of proteins into ER membrane 4. Protein folding and processing in ER 5. Quality control in ER
6. Export of proteins from ER 6.1 vesicle formation
a) COP II coat structure, assembly and disassembly 6. 2 Vesicle fusion
6.3 Trafficking pathways 6.4 Retrieval pathways
6.5 Unconventional pathways
Role Name Affiliation
Subject Coordinator <Dr. Sujata Bhargava>
Paper Coordinator <Dr. Nutan Malpathak>
Content Writer/Author (CW) <Dr. Gauri Abhyankar>
Content Reviewer (CR) < Dr. Nutan Malpathak>
Language Editor (LE) < Dr. Nutan Malpathak>
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1. Introduction:
Eukaryotic cells have very complex internal organization. So sorting and targeting of proteins to their correct destination is a big and challenging task. Protein sorting starts during translation. Different proteins are synthesized on ribosomes bound to ER. As translation proceeds, the polypeptide chains are transported to ER. These proteins are properly folded and processed in ER. They are then transported to other organelles.
Secretory proteins follow a definite path, called as secretory pathway. It starts with rough ER. Secretory proteins are then transported to golgi, to secretory vesicles to cell exterior. In eukaryotic cells, proteins that are meant for secretion or those that are going to be moved to ER, golgi, lysosomes or plasma membrane are initially directed at ER. These proteins are translated on membrane bound ribosomes. Those proteins which are going to remain in cytoplasm or will be transported to nucleus, mitochondria, chloroplast or peroxisomes are synthesized on free ribosomes.
3. Targeting proteins to ER –
In higher eukaryotes, proteins enter ER co-translationally (during their synthesis), whereas, yeast shows both co-translational as well as post-translational entry of proteins in ER (Fig 1).
The first step in co-translational pathway is identifying secretory proteins and directing them to ER. The ER obtains selected proteins from the cytosol as they are being synthesized. These proteins are broadly classified in two types:
a) Transmembrane proteins - they are partly moved across the ER membrane. These proteins become embedded in the ER membrane itself.
b) Water soluble proteins - these are fully translocated across the ER membrane and are released into the ER lumen.
Some of the transmembrane proteins function in the ER, but many are meant to go the plasma membrane or the membrane of another organelle. The water-soluble proteins would either go for secretion or remain in the lumen of ER (Alberts et al, 2002).
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Fig 1: Co-translational and posttranslational protein translocation. (A) Ribosomes bind to the ER membrane during co-translational translocation. (B) By contrast, ribosomes complete the synthesis of a protein and release it prior to post-translational translocation (Alberts et al, Molecular Biology of the cell, 2002).
The growing polypeptide chain of secreted proteins contains a signal sequence at the amino terminus (N – terminus). Signal sequences are short stretches of hydrophobic amino acids, about 20 – 25 amino acids long. They show large variation in their amino acid sequence, but irrespective of the variation, each signal sequence has eight or more nonpolar amino acids at its center. These signal sequences are recognized by a signal recognition particle (SRP).
SRP consists of six polypeptides and a small cytoplasmic RNA. The crystal
Fig 2: The signal-recognition particle (SRP). (A) SRP is a rod-like complex containing six protein subunits and one RNA molecule (shown in red). The SRP RNA forms the backbone that links the domain of the SRP containing the signal sequence binding pocket to the domain responsible for pausing translation. A bound
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signal sequence is shown as a green helix. (B) SRP bound to the ribsosome visualized by cryo-electron microscopy (Alberts et al, Molecular Biology of the cell, 2002).
structure of the SRP protein shows signal sequence binding site. It is a large hydrophobic pocket lined by methionines. The SRP is a rod-like structure. Its one end binds to the ER signal sequence while the other end blocks the elongation factor binding site at the interface between the large and small ribosomal subunits (Fig 2). This block halts protein synthesis as soon as the signal peptide comes out from the ribosome. This small pause gives enough time to the ribosome to bind to the ER membrane before completion of the polypeptide chain. By doing this it ensures that the protein is not released into the cytosol as well as it takes care that the protein folding will not occur before the polypeptide chain moves in the ER lumen. Once the SRP–ribosome complex is formed, it binds to the SRP receptor present on ER membrane. This interaction brings the SRP–ribosome complex to a protein translocator known as translocon. The SRP and SRP receptor are then released, ribosome binds to translocon. Signal sequence is inserted into the translocon. Translation resumes and the translocon shifts the growing polypeptide chain across the membrane into the ER lumen (Fig 3). When the protein is transported to ER lumen, the signal sequence is chopped off or removed by signal peptidases present in the ER lumen.
Functioning of SRP and SRP receptor requires binding of GTP. Hydrolysis of GTP to form GDP brings about dissociation of SRP.
Fig 3: The SRP binds to both the exposed ER signal sequence and the ribosome and causes a pause in translation. The SRP receptor in the ER membrane binds the SRP–ribosome complex and directs it to the translocator. The SRP and SRP receptor are then released, leaving the ribosome bound to the translocator in the ER membrane. The translocator then inserts the polypeptide chain into the membrane and transfers it across the lipid bilayer. The translocator is closed until the ribosome has bound, so that the permeability barrier of the ER membrane is maintained at all times (Alberts et al, Molecular Biology of the cell, 2002)
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The core of the translocator is called Sec61 complex (Fig 4).
Fig 4: Structure of the Sec61 complex. (A) A top view of the Sec61 complex of the archae Methanococcus jannaschii seen from the cytosol side of the membrane. The Sec61a subunit is shown in blue and red; the two smaller b- and g-subunits are shown in gray. The yellow short helix forms a plug that seals the pore when the translocator is closed. To open, the complex rearranges itself to move the plug helix out of the way.
(B) Model of the closed and open states of the translocator are shown, illustrating how a signal sequence could be released into the membrane after opening of the seam (Alberts et al, Molecular Biology of the cell, 2002).
It constitutes of three subunits that are highly conserved from bacteria to eukaryotic cells. The pore is gated by a short helix. It opens only transiently when a polypeptide chain traverses the membrane. The pore is kept closed In an idle translocator, so that the membrane remains impermeable to ions, such as Ca2+, which otherwise would leak out of the ER. The structure of the Sec61 complex is such that the pore can also open along a seam on its side. This opening allows lateral access to a translocating peptide chain into the hydrophobic core of the membrane. This process is necessary for release of a cleaved signal peptide into the membrane and for the integration of membrane proteins into the ER membrane (Fig 5).
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Fig 5: A model to explain how a soluble protein is translocated across the ER membrane. On binding an ER signal sequence, the translocator opens its pore, allowing the transfer of the polypeptide chain across the lipid bilayer as a loop. After the protein has been completely translocated, the pore closes, but the translocator now opens laterally within the lipid bilayer, allowing the hydrophobic signal sequence to diffuse into the bilayer, where it is rapidly degraded (Alberts et al, Molecular Biology of the cell, 2002).
Large number of proteins in yeast are moved to ER after they complete their translation (post translational targeting). These proteins are synthesized on free ribosomes present in cytoplasm. For transport of these proteins SRP is not required. They have a signal sequence which is recognized by a specialized receptor protein known as Sec62 / Sec63 complex. This complex is associated with translocon in ER membrane.
Post translational movement of proteins to ER is also dependent on cytosolic HSP70 chaperones. They maintain polypeptide chain in an unfolded form, so that it can enter the translocon. Another HSP70 chaperone, called as BiP, is present within the ER lumen. It pulls the protein from the translocon into the ER.
4. Insertion of proteins into ER membrane –
Secretory proteins are delivered to ER lumen by movement through ER membrane.
But some proteins are not destined as secretory proteins but they need to be incorporated in plasma membrane or in membranes or other organelles. Such proteins do not reach ER lumen. Instead, they are first incorporated into ER membrane. From there, they move to other organelles along the same secretory pathway. They are not transported as secretory
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proteins but they move along secretory pathway as membrane components or integral membrane proteins.
Integral membrane proteins are inserted in membrane by small membrane spanning portion. They typically are hydrophobic sequences of α helices consisting of 20 – 25 amino acids. But insertion of various membrane proteins differs (Fig 6).
Fig 6: Orientations of membrane proteins - The two proteins at left and center each span the membrane once, but they differ in whether the amino (N) or carboxy (C) terminus is on the cytosolic side. On the right is an example of a protein that has multiple membrane-spanning regions (Cooper & Hausmann, The Cell:A molecular Approach, 2009).
There are many different ways of protein insertion into the ER membrane.
a) Proteins have signal sequence at N-terminus, so their carboxy terminus is exposed to cytoplasm and N-terminus enters in ER lumen. They are then anchored in membrane by other α helix present in the middle of the protein. This sequence is called as stop- transfer sequence. It signals change in conformation of translocon channel and stops further movement of polypeptide chain across ER membrane. Then the translocon subunits separate and the protein is anchored in the membrane (Fig 7).
b) Some proteins have internal signal sequence. Such proteins are not cleaved by signal peptidases but they are recognized by SRP and are anchored to the ER membrane.
These signal sequences work as transmembrane α helices, so they exit translocon and anchor proteins to the membrane. Depending on the orientation of signal
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sequence proteins anchored in ER membrane can have their N-terminus or C- terminus exposed to cytosol (Fig 7).
Fig 7: An internal ER signal sequence binds to the translocator in one of two different ways, leading to a membrane protein that has either its C-terminus (pathway A) or its N-terminus (pathway B) in the ER lumen:
if there are more positively charged amino acids immediately preceding the hydrophobic core of the start- transfer sequence than there are following it, the membrane protein is inserted with their C-terminus, whereas, if there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer sequence than there are preceding it, the membrane protein is inserted with its N-terminus (Alberts et al, Molecular Biology of the cell, 2002).
c) Some proteins traverse the membrane many times. These proteins usually contain multiple alternating internal signal sequence and transmembrane stop-transfer sequence (Fig 8).
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Fig 8: In this protein, an internal ER signal sequence acts as a start-transfer signal and initiates the transfer of the C-terminal part of the protein. At some point after a stop-transfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane. (B) The hydrophobic region nearest the N-terminus serves as a start-transfer sequence that causes the preceding N-terminal portion of the protein to pass across the ER membrane (C) The final integrated protein has its N-terminus located in the ER lumen and its C-terminus located in the cytosol. The blue hexagons represent covalently attached oligosaccharides. Arrows indicate the paired start and stop signals inserted into the translocator (Alberts et al, Molecular Biology of the cell, 2002).
5. Protein folding and processing in ER –
Polypeptide chains need to be folded correctly such that they attain their 3 dimensional conformations. This is very crucial for their proper function. For secretory proteins, ER is an important site for many processes viz. – protein folding, multi-subunit protein assembly, formation of disulphide bonds, glycosylation and addition of glycolipid anchors to plasma membrane proteins.
a) Multi-subunit protein formation – Proteins move to ER lumen as unfolded chains of polypeptides. Folding of these polypeptides to attain proper 3 dimensional
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structure is facilitated by molecular chaperones like BiP. It binds to unfolded polypeptide chain and helps in assembly of multi-subunit proteins.
b) Formation of disulphide bonds – Protein folding and assembly requires disulphide bond formation between side chains of cysteine because it helps stabilize the tertiary and quaternary structure of many proteins. These bonds do not form in cytoplasm because bond formation needs oxidizing environment that is present in ER lumen. The bond formation is carried out by enzyme – protein disulphide isomearse, located in ER lumen which catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S–S) bonds.
c) Glycosylation –N-linked glycosylation takes place in ER on specific asparagines residues (Fig 9). Oligosaccharide units are added to acceptor asparagines residues present in the polypeptide chain. Oligosaccharides consist of 14 sugar residues and are synthesized on lipid carrier – dolichol. This dolichol is rooted in ER membrane.
The oligosaccharides along with sugar residues are transferred from dolichol to asparagine as a single unit. This transfer is very specific. It happens at a specific consensus sequence – Asn – X – Ser/Thr – by a enzyme called oligosaccharyl transferase. One copy of oligosaccharyl transferase is associated with each protein translocator. It permits the enzyme to scan and glycosylate the incoming polypeptide chains efficiently. The precursor oligosaccharide is linked to the dolichol lipid by a high energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction. While still in the ER, three glucoses and one mannose are quickly removed from the oligosaccharides of most glycoproteins.
This oligosaccharide trimming continues further in the Golgi apparatus.
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Fig 9: Protein glycosylation in the rough ER. Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated on target asparagines amino acids. The precursor oligosaccharide is transferred to the asparagine as an intact Unit (Alberts et al, Molecular Biology of the cell, 2002).
d) Glycolipid anchor formation – Some proteins are attached to plasma membrane by glycolipids. They are called glycosyl phosphtidyl inositol anchors (GPI anchors) and they are produced in ER membrane. Once protein synthesis completes, the anchors are added to C-terminus of certain proteins. Protein is cleaved at C- terminus, the polypeptide chain is the attached to GPI anchor in such a way that the protein remains attached to membrane only through its associated glycolipid (Fig 10). Then they are transported as membrane components to cell surface through secretory pathway.
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Fig 10: Addition of GPI anchors (Alberts et al, Molecular Biology of the cell, 2002).
6. Quality control in ER –
Cells carefully monitor the amount of misfolded protein in various compartments. A fairly large number of proteins in ER are degraded mainly because they cannot fold properly. So ER identifies mis-folded proteins and makes them go for degradation (Brandizzi et al, 2003). ER quality control involves atleast 4 chaperones, protein disulphide isomerase and supporting proteins. Two chaperones viz calnexin and calreticulin help glycoproteins to fold correctly (Fig 11). They help folding by binding sugar residues on partially folded glycoproteins before complete translocation. The chaperone allows the glycoprotein to go through multiple cycles of folding so that it can attain correct conformation. Even then if the protein is misfolded, the chaperone sends the protein back to cytoplasm through retro-translocon. When something comes through retro-translocon in the cytoplasm, it will be marked by ubiquitination and degraded in proteasome (Fig 12).
ER-resident proteins are retained in the ER or are recycled back from the Golgi apparatus (Vitale & Denecke 1999). Markers for the ER include GFP-HDEL fused to an N- terminal signal peptide and resident proteins such as the SAR1 GDP/GTP exchange factor SEC12, the luminal chaperone BiP, and the calcium-binding proteins calreticulin and calnexin (Irons et al. 2003).
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Fig 11: The role of N-linked glycosylation in ER protein folding. The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. Removal of the terminal glucose by a glucosidase releases the protein from calnexin. A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble ER resident protein (Alberts et al, Molecular Biology of the cell, 2002).
Fig 12: The export and degradation of misfolded ER proteins. Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. Misfolded membrane proteins follow a similar pathway (Alberts et al, Molecular Biology of the cell, 2002).
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Many times when misfolded proteins accumulate in ER, they show unfolded protein response (UPR). It involves many specific effects like an increased transcription of genes encoding - ER chaperones, proteins involved in retrotranslocation and protein degradation in the cytosol, and many other proteins that help to increase the protein folding capacity of the ER. Misfolded proteins activate a second transmembrane kinase in the ER that phosphorylates a translation initiation factor and inhibits it. Therefore, there is reduction in production of new proteins throughout the cell. The reduction in translation also helps to reduce the flux of proteins into the ER, and checks the load of proteins that need to be folded there. Finally, a third gene regulatory protein is initially synthesized as an integral ER membrane protein. When misfolded proteins accumulate in the ER, the transmembrane protein is transported to the Golgi apparatus, where it encounters proteases that cleave off its cytosolic domain, which can now migrate to the nucleus and help activate the transcription of the genes encoding proteins involved in the unfolded protein response (Fig 13).
Fig 13: Unfolded protein response (Alberts et al, Molecular Biology of the cell, 2002).
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7. Export of proteins from ER –
Newly synthesized proteins cross the ER membrane to enter the biosynthetic- secretory pathway. During their subsequent transport, these proteins are successively modified as they pass through different organelles. It is very crucial to keep the balance between forward and backward (retrieval) during the secretory pathway. Some transport vesicles move certain cargo molecules from one compartment to the next, while others retrieve specific proteins and return them to a previous compartment.
7.1 Vesicle formation –
Proteins that are present in ER and are destined for the Golgi apparatus are packaged into small COPII-coated transport vesicles (Fig 14). These vesicles are formed from ERES. Proteins that are to leave ER and go to some other organelle selectively enter the transport vesicles and they are concentrated in these vesicles.
For vesicle formation, a small GTPase is activated by its GDP/GTP exchange factor. The activation results in recruitment of coat proteins.
Fig 14: The recruitment of cargo molecules in to ER
(http://images.slideplayer.com/13/4089230/slides/slide_80.jpg).
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a) COP II coat structure, assembly and disassembly –
COPII coat proteins employment needs SAR1 GTPase and its GDP/GTP exchange factor. It is type II trans-membrane protein SEC12 which is present in ER. Arabidopsis encodes three SAR1 GTPases and two SEC12 proteins (Vernoud et al. 2003).
The sequence of events in COPII subunit assembly has been worked out in some detail (Kuehn et al, 1998). COPII budding in plants requires only Sar1-GTP, Sec23p/24p and Sec13/31. The first step is activation of Sar1p. It is brought about by membrane-bound Sec12p. It catalyses GDP–GTP exchange on Sar1p and activates it. Once activated, it starts off the recruitment of the various components of the COPII complex on to specific budding sites on ER membranes. Following this activation, the Sec23p/24p complex interacts with Sar1p-GTP, gets recruited and forms a functional pre-budding complex. This complex binds to Sar1p and starts collecting cargo proteins at the budding site. It then adds Sec13p/31p which forms an outer shell complex (Fig 15). This Sec13p/31p probably acts like clathrin that polymerizes at the membrane surface of the budding site and physically drives the curvature of membrane to generate a bud (Tang et al, 2005). The disassembly of the COPII coat is triggered by the hydrolysis of the GTP on Sar1p.
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Fig 15: Schematic diagram illustrating COPII-mediated exit from the endoplasmic reticulum (ER) for transport to the Golgi. [1] The box represents a schematic view of a COPII-coated membrane region. The red structure represents putative scaffold proteins, the green bar a typical membrane cargo (e.g. a SNARE molecule), the light blue bar a soluble cargo receptor and the pink oval a soluble luminal cargo protein [2].
two possible non-mutually exclusive models: COPII vesicles coalesce to form or fuse with existing EGTCs, or the latter being derived by direct fission of ER tubules/sacs (black arrows indicate directionality) [3] The nature of the EGTCs is enigmatic. They may have different types of cargo and may represent an EGTC at different stages of maturation (denoted by the gradations of pink). Some EGTCs have distinct budding profiles (as indicated by the magnified schematic view) which may represent COPI-mediated retrograde transport in action within these structures, a plausible mechanism for maturation [4]. How EGTCs, especially those generated by peripheral ERES, at a distance away from the Golgi, engage motor proteins and travel to the Golgi stack along microtubule tracks (dotted line arrows) is not clearly known in detail (Tang et al, Biochimica et Biophysica Acta 1744: 293– 303, 2005).
These cargo proteins exhibit a transport exit signals on their cytosolic surface. The signal is recognized by the COPII coat. These coat components act as cargo receptors and are recycled back to the ER after they have delivered their cargo to the Golgi apparatus. Soluble cargo proteins in the ER lumen have exit signals that attach them to transmembrane cargo receptors. These receptors bind through exit signals to components of the COPII coat. At a lower rate, proteins without exit signals can also enter transport vesicles, so that even ER resident proteins slowly leak out of the ER and are delivered to the Golgi apparatus. Similarly, secretory proteins that are made in high concentrations may leave the ER without the help of exit signals or cargo receptors. Only properly folded proteins and completely assembled multimeric protein complexes can leave ER in transport vesicles.
6.2 Vesicle Fusion –
Vesicle fusion with the target membrane occurs after the vesicle has shed its coat.
Initially, a Rab GTPase on the vesicle membrane interacts with a tethering protein complex on the target membrane. Then SNARE proteins residing on the opposite membranes form a trans-complex, which results in membrane fusion.
Rab GTPases and effectors –
Rab GTPases and their effector proteins tether vesicles to target membranes (Whyte &
Munro 2002). Arabidopsis encodes 57 Rab GTPases grouped into eight subfamilies, A–H (Vernoud et al. 2003). However, only a few plant Rab GTPases have been functionally characterized. Several tethering complexes studied in yeast and animals have putative homologs in Arabidopsis, including the exocyst at the plasma membrane, TRAPP at the cis-Golgi, VFT/GARP at the trans-Golgi, C-VPS at the vacuolar membrane, and several
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components of the Vps34/COG complex involved in Golgi retrograde transport (Juergens
& Geldner 2002, Elias et al. 2003).
SNAREs and associated proteins –
SNARE proteins initiate membrane fusion by forming a trans-complex via their distinct R- or Q-SNARE motifs (Jahn et al. 2003). A R-SNARE on the vesicle pairs up with two or three Q-SNAREs on the target membrane. Each component of a SNARE complex is a member of a protein family, and different SNARE complexes are involved in different trafficking pathways. Arabidopsis encodes at least 54 SNAREs. Following membrane fusion, α-SNAP and the N-ethylmaleimide-sensitive factor (NSF) assist in the disassembly of cis-SNARE complexes (Jahn et al. 2003).
Once the transport vesicles separate from ER exit sites, they shed their coat and start to fuse with one another. This fusion of membranes from the same compartment is called homotypic fusion, whereas, membrane fusion from two different organelles is called heterotypic fusion. When ER-derived vesicles fuse with one another they form vesicular tubular clusters. They have a convoluted appearance when observed through the electron microscope (Fig 16).
Fig 16: An electron micrograph of vesicular tubular clusters forming from ER membrane (Alberts et al, Molecular Biology of the cell, 2002).
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These clusters represent a new compartment that is separate from the ER and lacks many of the proteins functional in ER. They are continuously generated and function as transport containers. They clusters are relatively short-lived because they move quickly along microtubules to the Golgi apparatus. Immediately after their formation, they begin to bud off transport vesicles of their own. These vesicles are COPI-coated. They bring back resident proteins and cargo receptors to ER. The COPI coat assembly begins almost immediately after the COPII coats have been shed. The retrograde transport continues as the vesicular tubular clusters move towards the Golgi apparatus.
6.3 Trafficking pathways –
A secretory default pathway leads from the ER via Golgi stacks to the plasma membrane (Figure 17). Storage proteins can bypass the Golgi altogether or exit from the cis-Golgi en route to storage vacuoles via intermediate compartments. It has been shown that soluble proteins are exported from the ER in plants by a COPII-dependent bulk flow mechanism (Phillipson et al, 2001).
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Fig 17: Simplified diagram of plant endomembrane system and trafficking pathways. Secretory cargo is transported from the endoplasmic reticulum (ER) via the Golgi/TGN to the plasma membrane (PM). Cargo destined for the lytic vacuole (LV) is sorted in a BP-80 dependent fashion into clathrin-coated vesicles (CCV) at the TGN and transported to the prevacuolar compartment (PVC/MVB). Cargo destined for the protein- storage vacuole (PSV) is trafficked from the ER to intermediate compartments (DV, PAC, MVB/DIP), from the cis-Golgi via dense vesicles (DV), and from the trans-Golgi via clathrin-coated vesicles (CCV). PSV and LV may fuse to give a large central vacuole (CV). The endocytic pathway involves an early/sorting endosome (EN) for recycling of plasma-membrane proteins and a multivesiculate late endosome corresponding to the PVC. Vacuolar markers, cargo receptors (ERD2, BP-80, PV72), coat proteins (COPI, COPII, AP-1, AP-2), SAR1, ARF, and Rab GTPases, and syntaxins (SYP) are indicated. DIP, DIP organelle; Endo, endocytosis;
Exo, exocytosis; MVB, multivesicular body; PAC, precursor-accumulating compartment (Juergens, Annu.
Rev. Cell Dev. Biol. 20:481–504, 2004).
Transport mediated by COPI and COPII is very similar in many respects. COPII carriers originate in the ER and export newly synthesized proteins to the Golgi complex, whereas, COPI vesicles bud from the cis-Golgi and travel to the ER.
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Export from the ER to the Golgi occurs via a COPII mediated mechanism but evidence for a second anterograde pathway independent of COPII has also been postulated (Toermaekangas et al, 2001).To balance these transport pathways, at least one retrograde pathway should be there to carry cargo molecules back from the Golgi to the ER. Retrieval of proteins from Golgi to ER is very much necessary. Some proteins are ER resident proteins that may have a role in the folding and modification of newly synthesized proteins. Proteins involved in the export machinery itself may also be salvaged for their reuse in a subsequent round of vesicle formation and transport. The retrieval mechanism is based on the presence of signals in the protein sequence. It is recognized by a receptor or receptor-like protein. The binding of ligand to receptor induces the formation of a retrograde vesicle to transport ER-resident proteins back to their destination (Hanton et al, 2005).
Relatively high mobility of the ER and Golgi may pose certain challenges in transport mechanism. This might have led to the evolution of specific mechanisms in plants so that efficient trafficking can occur. Three possible mechanisms for protein transport between the ER and the Golgi have been postulated based on the mobility of the early plant secretory pathway (Fig 18).
a) Vacuum cleaner model – Golgi bodies move along the ER to reach vesicles budding from ERES. Cargo molecules can be transferred from ERES to the Golgi.
b) Stop-and-go model – It suggests that there is an unidentified signal present on fixed ERES that causes Golgi bodies to become transiently detached from the actin microfilaments, while they acquire cargo proteins from the ER. Later, they reattach to the actin and continue to move.
c) The mobile ERES model – Golgi bodies and ERES move together, allowing continual protein transport between the two organelles.
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Fig 18: Comparison of the models for protein transport between ERES and Golgi bodies. A) The vacuum cleaner model. B) The stop-and-go model. C) The mobile ERES model. (Hanton et al, Traffic 6:267 – 277, 2005).
6.4 Retrieval pathways –
The retrieval pathway depends on ER retrieval signals. So resident ER membrane proteins contain signals binding directly to COPI coats and so get packaged into CoPl- coated vesicles for retrograde delivery to the ER. The best-characterized retrieval signal consists of two lysines, followed by any two other amino acids, at the extreme c-terminal end of the ER membrane protein. It is called a KKXX sequence. Soluble ER resident proteins, such as BiP also contain a short retrieval signal at their C-terminal end, but contains - Lys-Asp-Glu-Leu - (KDEL sequence). Soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor. It is a multi-pass transmembrane protein. It binds to KDEL sequence and packages the protein displaying this sequence into coPl-coated vesicles (Fig 19). Most membrane proteins that function at the interface between the ER and Golgi apparatus, including v- and t-sNAREs and some cargo receptors, enter the retrieval pathway back to the ER. Presence of retrieval signals increase the efficiency of retrieval process. Many Golgi enzymes cycle constantly between the ER and the Golgi, but their rate of return to the ER is slow enough for most of the protein to be found in the Golgi apparatus.
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Fig 19: A model for the retrieval of soluble ER resident proteins. ER resident proteins that escape from the ER are returned b y vesicular transport. (A)The KDEL receptor resent in vesicular tubular clusters and the Golgi apparatus captures the soluble ER resident proteins and carries them in COP|-coated transport vesicles back to ER. Upon binding its ligands in this environment, the KDEL receptor may change conformation so as to facilitate its recruitment in to budding COPI-coated vesicles(.B )The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi
Apparatus (Alberts et al, Molecular Biology of the cell, 2002).
6.5 Unconventional pathways –
We know that vacuolar proteins reach the vacuole through Golgi using secretory vesicles. Generally those pathways that start from the ER and reach the vacuole bypassing the Golgi complex have been reported as exceptions to the classical Golgi path.
These alternative transport mechanisms are usually activated in particular cellular situations. In plant cells, the existence of alternative pathways to the main vacuolar sorting mechanism is well documented (Herman & Schmidt, 2004). E.g. ER bodies are directly transported to the vacuole. These ER bodies in many cases are larger in diameter than the secretory vesicles. Two main types of proteins – storage proteins and enzymes – have been observed to follow this route. Storage proteins form spherical aggregates ranging from 0.2 to 1.8 μm in size and are detected mainly in cereal seed tissues. These aggregates are formed during storage protein accumulation. Seed storage proteins can be transported directly from the ER to protein storage vacuoles (PSVs), apparently by atypical autophagic processes, e.g. γ-gliadins in wheat endosperm (Levanomy et al, 1992).
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2. Brandizzi F, Hanton S, DaSilva LL, Boevink P, Evans D, et al. (2003) – ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants. Plant J. 34:269–81.
3. Cooper GM, Hausmann RE (2009) – The cell: A molecular Approach – ASM Press, Wasington.
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5. Hanton S, Bortolotti L, Renna L, Stefano G, Brandizzi F (2005) - Crossing the divide – Transport between the endoplasmic reticulum and golgi apparatus in plants.
Traffic 6:267 – 277.
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