DEVTOME.COM HOSTING COSTS HAVE BEGUN TO EXCEED 115$ MONTHLY. THE ADMINISTRATION IS NO LONGER ABLE TO HANDLE THE COST WITHOUT ASSISTANCE DUE TO THE RISING COST. THIS HAS BEEN OCCURRING FOR ALMOST A YEAR, BUT WE HAVE BEEN HANDLING IT FROM OUR OWN POCKETS. HOWEVER, WITH LITERALLY NO DONATIONS FOR THE PAST 2+ YEARS IT HAS DEPLETED THE BUDGET IN SHORT ORDER WITH THE INCREASE IN ACTIVITY ON THE SITE IN THE PAST 6 MONTHS. OUR CPU USAGE HAS BECOME TOO HIGH TO REMAIN ON A REASONABLE COSTING PLAN THAT WE COULD MAINTAIN. IF YOU WOULD LIKE TO SUPPORT THE DEVTOME PROJECT AND KEEP THE SITE UP/ALIVE PLEASE DONATE (EVEN IF ITS A SATOSHI) TO OUR DEVCOIN 1M4PCuMXvpWX6LHPkBEf3LJ2z1boZv4EQa OR OUR BTC WALLET 16eqEcqfw4zHUh2znvMcmRzGVwCn7CJLxR TO ALLOW US TO AFFORD THE HOSTING.

THE DEVCOIN AND DEVTOME PROJECTS ARE BOTH VERY IMPORTANT TO THE COMMUNITY. PLEASE CONTRIBUTE TO ITS FURTHER SUCCESS FOR ANOTHER 5 OR MORE YEARS!

Mechanisms of Protein Transport

The primary means of protein transport from the ER to the cis-Golgi is COPII-mediated vesicular transport. Failure of ER-to-Golgi transport of transmembrane proteins could result from a failure of such vesicular formation. A defect in the ER-associated guanine exchange factor (GEF) Sec12, any of the COPII coat proteins – Sec23/24, Sec13/31, or Sec16 – or the COPII-associated GTPase Sar1p could underlie such a failure. Various experimental techniques are available to distinguish between these various defects, and are summarized as follows.

Sec12 is a GEF which mediates the release of GDP from Sar1p, allowing Sar1p to bind GTP, causing a conformational change which exposes its anchor sequence and leads to its embedding in the ER lumen and serving as a nucleation point for coat formation. It is possible to test for a defect in either of these proteins using modifications of previously described techniques (Barlowe, 1993). First, microsomal membranes from the mutant strain are added to cytosol extracted from the same strain in vitro. High levels of WT-Sar1p-GTP should then be added to the solution in order to assess whether or not this restores vesicle formation using the same vesicle formation assay used by Barlowe et al. If vesicle formation is restored by WT-Sar1p-GTP this points to a defect in either the mutant-derived Sar1p or in the GEF responsible for its activation (Sec12). As such the functionality of the mutant-derived Sar1p should be assayed by purifying the protein from the mutant cells. This purified Sar1p should then be added to WT-microsomes and Sar1p-depleted cytosol derived from Sec12-overproducing mutant cells. If the purified Sar1p restores vesicular formation to Sar1p depleted cytosol (assessed using the abovementioned assay) then this suggests that it is not to blame for the defect in vesicular formation. If the mutant-derived Sar1p is still functional and added WT-Sar1p-GTP restores vesicle formation to these cells then this suggests a defect in the Sec12 protein resulting in a failure of the mutant cells to produce Sar1p-GTP.

Sec23 and Sec24 form a complex in vivo and associate with Sar1p-GTP and the cytosolic domain of transmembrane cargo proteins, respectively. Sec 13 and Sec31 also form a complex, which associates with ER-bound Sec23/24 complexes and facilitates membrane rounding and subsequent vesicular budding (Sato, 2004). Additionally, Sec16 has been shown to bind to Sec23/24/31 (Iinuma 2007). To assess the functionality of these proteins, they should first be immunoprecipitated from the cytoplasmic fraction of a lysate of this yeast strain using beads coated with an antibody specific to either Sec23 or Sec13. Precipitate should then be tested via Western blot in order to ascertain whether or not Sec24 co-immunoprecipitated with Sec23 and whether or not Sec31 co-immunoprecipitated with Sec13, and whether Sec16 precipitated with either of these proteins. If the complex forming functionality of these proteins is intact, they should be microinjected into yeast cells which have depleted of either Sec13 or Sec23 by means of siRNA in order to assess whether or not this restores vesicular formation to these cells (Townley, 2008). Vesicular formation in these cells can be monitored using an over-expressed fluorescently tagged secretory protein which will accumulate in the ER unless vesicle formation is restored by the addition of a functional form of the depleted protein. If these proteins successfully complex and restore budding capacity to depleted cells, they are not likely the cause of defective vesicular formation in the strain of interest.

While vesicular formation is a potential explanation for a failure of ER-to-Golgi transport, it is not the only explanation. Failures of vesicular fusion to the cis-Golgi would result in an accumulation of properly formed vesicles in the cytoplasm, unable to deliver their cargo proteins to the Golgi. Defects in the vesicle-associated Rab GTPase or in the SNARE proteins of either the transport vesicle or of the target vesicle could result in a failure of vesicular fusion, and additional experiments would be necessary to differentiate between these mechanisms.

Mechanisms of Vesicular Transport

The three primary classes of coated budding vesicles are clathrin-coated vesicles, COPI-coated vesicles, and COPII-coated vesicles. In general, budding vesicle (and tubule) formation initiates when a specific membrane-associated GTPase is activated and is bound by adaptor proteins which also bind specific cargo protein motifs. Adaptors associate with multimeric coat proteins which cross-link adaptor proteins, concentrating cargo proteins within the budding vesicle and generating membrane curvature, ultimately leading to vesicle budding and fission.

COPI-coated vesicles mediate intra-Golgi transport and retrograde Golgi-to-ER tranpsort of target proteins. The cytosolic Arf1 G protein is activated by a Golgi-associated Sec7 GEF, and Arf1-GTP binds the Golgi membrane. Arf1-GTP is then specifically recognized and bound by the COPI coatomer, a heptameric protein complex which contains both cargo-binding adaptor domains and cross-linking outer coat domains. Arf1-GTP/cargo-bound coatomer self assemble, binding other coatomer complexes to locally concentrate cargo and generate membrane curvature. Fission occurs following sufficient membrane curvature generation and is in part mediated by Arf-GAPS which induce Arf1-GDP release from the COPI vesicle.

COPII-coated vesicles mediate the anterograde transport of proteins from the ER to the Golgi. The cytosolic Sar1 GTPase initiates COPII coat recruitment when it is activated by the ER membrane-associated Sec12 GEF. Sar1-GTP inserts into the ER membrane and is bound by an adaptor protein complex consisting of Sec23/24, which bind Sar1-GTP and the cytosolic tails of transmembrane cargo proteins, respectively. Sec24 contains multiple domains which recognize and bind various ER-exit-associated amino acid motifs. Sec23/24 are then bound by the Sec13/ Sec31 outer coat complex, which binds multiple Sec23/24 subunits increasing local Sec23/24 concentration, thereby selecting for cargo while generating membrane curvature. As curvature increases the vesicle begins to bud and fission is dynamin-independent.

Clathrin-coated vesicles are involved in cargo-dependent receptor mediated endocytosis and in the sorting of trans-Golgi cargo proteins. Rather than associate with an active G protein, at the plasma membrane clathrin adaptor proteins bind the membrane-specific phosphoinositide PI(4,5)P2 to ensure proper membrane localization. These adaptors then associate with the cytosolic tails of cargo proteins or cargo receptors. Adaptor AP2, for example, binds to both PI(4,5)P2 and to cargo protein acidic dileucine motifs. Multiple cargo-bound adaptors are then bound by each triskelion clathrin protein, increasing local cargo concentration and generating membrane curvature via the formation of a geometric clathrin lattice. Vesicle fission is mediated by the dynamin GTPase which pinches the budding vesicle off of the membrane.

ER Quality Control Checkpoints

There are many mutations which would disrupt ER quality control mechanisms and cause a cell to secrete rather than degrade misfolded proteins. The two most commonly noted proteins which bind misfolded proteins and target them for retrotranslocation are calnexin and BiP, and a defect in either of these chaperones or of genes encoding proteins which regulate these chaperones would likely lead to increased secretion of misfolded proteins (Lodish, 2007). It has been proposed that calnexin binding of misfolded proteins and subsequent targeting of these proteins for degradation is enhanced by removal of multiple mannose residues from oligosaccharide chains; thus, defects in mannosidase activity may result in reduced retention of misfolded proteins by calnexin, and may thus result in increased secretion of these proteins (Liu, 1999). Alternatively there may be a mutation affecting the cellular machinery driving ER export, such as a defect in the cytoplasmic AAA ATPase p97 which may be responsible for pulling misfolded proteins from the ER into the cytoplasm (Lodish, 2007).

If the mutant cell line is a yeast mutant, one means to establish which mutations are causative of such a phenotype would be by means of a complementation study. This can be done by mating haploid cells of the mutant strain with haploid mutants of strains with known defects in proteins suspected to be defective in the mutant strain. For example, a strain expressing mutant calnexin has been found to be defective in ER-associated protein degradation (McCracken, 1996). If the diploid offspring still secretes abnormal levels of misfolded proteins, this indicates that the mutations did not complement and consequently suggests that a mutated gene in the known strain and a mutated gene in the novel strain code for the same protein.

A related technique relies upon the introduction of inducible promoters driving the over-expression of proteins suspected to be defective in these mutant cells. Yeast should be transformed with plasmids expressing the WT forms of ER proofreading-related proteins. Transformed strains should then be selected and screened for aberrant secretion of misfolded proteins before and after the induction of the inserted promoter. If the overexpression of a specific protein reduces secretion of misfolded proteins to background levels then this suggests that this protein was mutated in the strain secreting misfolded proteins and indicates that the WT form of this protein can restore a normal phenotype to these cells.

Sources

Barlowe C, d’Enfert C, Schekman R. Purification and Characterization of SAR1p, a Small GTP-binding Protein Required for Transport Vesicle Formation from the Endoplasmic Reticulum. J Biol Chem. 1993; 268(2): 873-879.

Iinuma T, et al. Mammalian Sec16/p250 Plays a Role in Membrane Traffic from the Endoplasmic Reticulum. J Biol Chem. 2007; 82: 17632-17639.

Liu Y, et al. Oligosaccharide Modification in the Early Secretory Pathway Directs the Selection of a Misfolded Glycoprotein for Degradation by the Proteaseome. J Biol Chem. 1999; 274(9): 5861-5867.

Lodish H, et al. Molecular and Cell Biology. New York, NY: WH Freeman and Company. 2008; p. 533-597.

McCracken AA, Brodsky JL. Assembly of ER-Associated Protein Degradation In Vitro: Dependence on Cytosol, Calnexin, and ATP. J Cell Biol. 1996; 132(3); 291-298.

Ogg SC, et al. A Functional GTPase Domain, but not its Transmembrane Domain, is Required for Function of the SRP Receptor β-subunit. J Cell Biol. 1998; 142(2): 341-354.

Sato K. COPII Coat Assembly and Selective Export from the Endoplasmic Reticulum. J Biochem. (2004) 136 (6): 755-760.

Siekevitz P, Palade GE. A Cytochemical Study on the Pancreas of the Guinea Pig V. In vivo Incorporation of Leucine-1-C14 into the Chymotrypsinogen of Various Cell Fractions. J Biophysic and Biochem Cytol. 1960; 7(4): 619-630.

Townley AK, et al. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J Cell Science. 121 (18): 3025. (2008)

Waters GM, Blobel G. Secretory Protein Translocation in a Yeast Cell-free System Can Occur Posttranslationally and Requires ATP Hydrolysis. J Cell Biol. 1986; 102: 1543-1550.


Science | Biology | Experiments


QR Code
QR Code mechanisms_of_protein_transport (generated for current page)
 

Advertise with Anonymous Ads