Molecular Biology of the Cell by Lodish et al The vast majority of proteins are synthesized on ribosomes in the cytosol and indeed many of these proteins remain within the cytosol. However, some proteins must leave the confines of the cytosol to exert their influence and it is the delivery of newly synthesized proteins to organelles such as the plasma membrane or the nucleus which is known as the "secretory pathway". Proteins are directed to their specific organelles by targeting sequences known as signal peptide sequences. These signal peptide sequences are on the N terminal end of the polypeptide chain. They all have a similar pattern comprising of three elements; i) a short, positively charged N terminal section, ii) a central hydrophobic region of between ten to fifteen amino acid residues-usually Leucine, Valine or Isoleucine- and iii) a short, more polar region -usually in the form of an alpha helix in non-polar environments- which is able to traverse the membrane of the endoplasmic reticulum. In the absence of these targeting sequences, proteins are released into the cytosol where they remain.
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Protein trafficking to the Endoplasmic Reticulum
http://life.lifesci.cn/upload/bio/ebook/secretory%20pathway.pdfThe targeting sequence on a given protein interacts with a specific corresponding receptor- the signal recognition particle, a 325-kD complex consisting of six different polypeptides with an RNA molecule. Binding of the signal sequence to the signal recognition particle results in the formation of an RNA-protein complex (ribonucleoprotein complex) in the cytoplasm. The signal recognition sequence then binds to the hydrophobic section of the signal peptide. This action blocks any further elongation of the polypeptide chain by blocking the ribosome. The ribosome migrates to the membrane of the endoplasmic reticulum carrying with it the SRP receptor and the attached docking proteins. The signal recognition particle attaches to this which positions the ribosome on the membrane.
The complex of the SRP and the ribosome diffuses to the surface of the rough endoplasmic reticulum where it can now bind with the SRP receptor and the translocon. The translocon is a multi-subunit assembly of both integral and peripheral membrane proteins which acts as a channel for the protein. The channel only opens upon binding of the translocon and the ribosome. Protein synthesis can now resume so the growing polypeptide chain is able to pass through the channel and into the lumen of the ER. The signal sequence is cleaved by a peptidase on the inner face of the membrane so a mature protein will never have a signal sequence. Soluble secreted proteins pass through the rough and then the smooth endoplasmic reticulum. From here they are ferried to the Golgi apparatus in transfer vesicles. The Golgi apparatus acts as a sorting station in which secreted proteins are packaged into secretory vesicles. These vesicles will fuse with the plasma membrane. Proteins of the plasma membrane travel all the way through the secretory pathway whereas proteins of the ER and Golgi are retained in their own organelles.
It is important to note that there is no direct physical connection between the endoplasmic reticulum and the Golgi apparatus or Golgi stacks. In order for proteins to move between these structures, they must be packaged into vesicles which are coated with special proteins known as "coat proteins" or COPs. Assembly of these "coats" is triggered by the binding of GTP to a small GTP-binding protein (GTPase) called ARF1p (ADP- ribosylation factor). GTPases are small intracellular switch proteins which bind and hydrolyze GTP. They have a number of important roles
Signal transduction of intracellular domain of trans-membrane receptors
Protein biosynthesis at the ribosomes
Control and differentiation during cell division
Translocation of proteins through the membrane
Transport of vesicles within the cell
Hydrolysis of GTP coupled to protein translocation
Molecular Cell Biology by Lodish et alIn order to understand how GTPases work, it is necessary to understand the complex structure of G proteins. There are two classes of GTPases: the G alpha subunit of heterotrimeric G proteins and monomeric G proteins. Heterotrimeric G proteins contain alpha, beta and gamma subunits. The alpha and gamma subunits are covalently attached by lipid molecules to the plasma membrane. The alpha subunit has an important role in GDP/GTP binding and has intrinsic GTPase activity. The alpha subunit binds to one side of the beta subunit and locks the GTPase domain in an inactive conformation which binds GDP. The gamma subunit binds to the opposite side of the beta subunit to form a single functional unit.
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The 'protein coats' surrounding these vesicles is disassembled by hydrolyzing GTP, allowing the uncoated vesicles to fuse with the membrane. There are three types of coated vesicles which transport cargo proteins from particular parent organelles to particular destination organelles; Clathrin, COPI and
Different coat proteins have different roles in trafficking vesicles
Molecular Biology of the Cell by Alberts et alCOPII. Vesicle fusion is controlled by SNARE proteins. There are two types of SNARE proteins: v- SNAREs are incorporated into transport vesicle membranes during budding, t-SNAREs are located in the membranes of target compartments. COPI vesicles are important in retrograde transport between the Golgi cisternae and from the cis- Golgi back to the rough ER. COPI vesicles bring them back to the ER where the v-SNARE proteins can be recycled. COPI forms a 'fuzzy' coat, in contrast to the polyhedral coat formed by Clathrin around vesicles. COPII coated vesicles participate in anterograde transport from the rough ER to the Golgi and they contain newly synthesized proteins destined for the Golgi, cell surface or lysosomes They also contain vesicle components such as v-Snares which target vesicles to the cis-Golgi membrane. The coat of a COPII vesicle contains two conserved protein heterodimers. The ARF1p GTPase is associated with both CopI and Clathrin coated vesicles. In the case of COPII coated vesicles, a GTPase which is very similar to ARF1p is present known as Sar1.
ARF and Sar1 play a pivotal role in the polymerization and de-polymerisation of coat proteins on a vesicle. The assembly and disassembly of the coat proteins is coupled to Sar1p-GTP hydrolysis. The COPII coat proteins are necessary for the capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum. The first step in the assembly of a COPII coat requires a type II integral protein which is present in the membrane of the endoplasmic reticulum, Sec 12. Sec12 is the guanine nucleotide exchange factor for Sar1 so it causes recruitment of Sar1 to a vesicle formation site on the membrane of the endoplasmic reticulum. Sec 12 is strictly localized to the membrane of the endoplasmic reticulum so COPII assembly is restricted to this area. It recruits Sar1 by causing the release of GDP for Sar1-GTP on the cytosolic face of the membrane, allowing GTP to bind to Sar1. Once GTP is bound to Sar1, its hydrophobic N terminus becomes exposed as the binding of GTP causes a conformational change, embedding it in the ER membrane. Sar1 can now acts as a binding site for two heterodimeric sub complexes Sec23/Sec24 and Sec13/Sec31. Binding of Sec13p/Sec31p enhances the GAP activity of Sec23p on Sar1p. The capture of cargo and SNARE proteins occurs during the binding of these sub complexes to Sar1. Sec 23/24 associates with the v-SNARE or t-SNARE complexes forming a complex which is not destabilized during hydrolysis of GTP on Sar1p.
A completely different set of GTPases (Rab proteins) control the docking of vesicles to their target membranes. These Rab proteins are members of the same GTPase super family as ARF1, Ras and Sarp1 so they interconvert between its active (GTP bound) and inactive (GDP bound) forms in the same way when acted upon by a guanine nucleotide exchange factor. When the Rab protein is converted from its GDP bound state to its GTP bound state, a conformational change occurs exposing a lipid group which is covalently attached to the Rab protein. This lipid group anchors the Rab protein to the membrane of the
Rab protein and its role in vesicle docking
Molecular Biology of the cell by Alberts et al transport vesicle. The Rab protein, in its GTP state, remains bound to the surface of the transport vesicle and it then binds to Rab effector proteins which are present on the target membrane. These effector proteins help the vesicle to dock and also play a pivotal role in the pairing of v-SNAREs and t-SNAREs. The Rab protein then hydrolyses its bound GTP causing the release of the Rab-GDP complex into the cytosol where it can be recycled when needed.
The secretory pathway is a highly regulated, complex series of interactions between enzymes, coat complexes and GTPases. Correct navigation through the complex tracks of the secretory pathway ensures that only proteins which have been correctly modified and folded will end up at their intended destination. Through initial recognition of signal sequences through to interactions with Rab proteins, vesicles transporting vital components for the cells make their way from the cytosol to the cell surface. Misfolded proteins are retained in the endoplasmic reticulum by chaperones which block exit signals and sometimes even anchor these faulty proteins in the cytosol of the Endoplasmic Reticulum. These misfolded or faulty proteins are transported back into the cytosol and will eventually be degraded by proteasomes. Thus, the complex secretory pathway and its control by GTPases can be seen as a sort of "safety net", preventing proteins which may be faulty interfering with the functions of normal proteins which have been implicated in conditions such as Alzheimer's.
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