Recent work also suggests that retrograde transport can be used to degrade misfolded membrane proteins. CUP-2 is one of two worm Derlin homologs (). Derlin proteins localize mainly to the ER membrane and function in ER-associated degradation (ERAD) in mammalian and yeast systems. In mutants, the unfolded protein response (UPR) is activated, suggesting accumulation of ERAD substrates in the ER (). In addition to this ER-associated phenotype, mutants abnormally accumulate CUP-7/MCA-3 on the plasma membrane. Similar accumulation of transmembrane proteins on the plasma membrane was observed when Derlin-1 was knocked down in RAW264.7 macrophage cells. Furthermore, when misfolding of surface transmembrane proteins was artificially induced in the RAW264.7 cells, misfolded surface membrane proteins were degraded in a Derlin-1- and proteasome-dependent manner (). CUP-2 and Derlin-1 seem to localize to endosomes in addition to the ER. These observations imply that CUP-2 and Derlin-1 are involved in the quality control of transmembrane proteins at the plasma membrane, or in endocytic compartments (). It was further reported that CUP-2 physically interacts with SNX-1, a subunit of the retromer complex, potentially explaining how CUP-2 is retrieved from endosomes to the Golgi (). It was proposed that CUP-2/Derlin would further transport misfolded cargo to the ER and retranslocate it to the cytoplasm for proteasome-dependent degradation (; ).
ESCRT-I, -II, and -III are thought to be required not only for sorting and concentration of cargos into ILVs, but also for the formation of the ILVs. ESCRT-II binds to VPS-20 of ESCRT-III, and this complex binds specifically to highly curved membranes on synthetic liposomes , suggesting that it senses the curvature of lipid bilayer (). VPS-32 of ESCRT-III also binds to liposomes and distributes as individual particles throughout the lipid bilayer. When VPS-32 is incubated with ESCRT-II and VPS-20, it forms filamentous structures, which specifically bind to the edges of highly curved membranes. In the absence of protein, some gaps occured on the flat supported lipid bilayer and filled with membrane over time. In the presence of ESCRT proteins, such gaps on the supported lipid bilayer are closed more rapidly and uniformly, suggesting that ESCRT-III promotes remodeling of the lipid bilayer (). Recent studies have shown that ESCRT-I and ESCRT-II, in combination, deform the membrane into buds. ESCRT-III cleaves the buds to generate ILVs when incubated with giant unilamellar vesicles (; ). Assembly of ESCRT-II with VPS-20 specifically on the highly curved membrane of endosomes may be required for nucleating ESCRT-III filaments on endosomes to generate ILVs.
Translation of mRNA into a protein requires ribosomes, mRNA, tRNA, exogenous protein factors and energy in the form of ATP and GTP. Translation occurs in three major steps: initiation, elongation and termination.
During elongation the protein is synthesized one amino acid at a time on the 80S ribosome. This process occurs in three major steps: binding of charged tRNA, peptide bond formation, translocation of the growing peptide chain.
All tRNA's are similar in structure. The TC arm participates in binding of the charged tRNA to a site on the ribosome where protein synthesis occurs. The DHU (or D) arm is necessary for recognition by the proper aminoacyl tRNA synthase (the enzyme). The acceptor end is at the 3' terminus and ends in the sequence CCA. The anticodon arm consists of seven nucleotides, the sequence of which is read 3' to 5' (opposite convention to the usual 5' to 3').
When a stop codon appears at the translation is terminated. There are no tRNA's that recognize stop codons. Instead proteins called releasing factors, eRF, recognize the stop codon. The releasing factors along with peptidyl transferases and GTP catalyze the hydrolysis of the bond between the polypeptide chain and the tRNA. The protein and tRNA disassociate from the site and the ribosome dissociates into the 40S and 60S subunits releasing the mRNA.
The rate of protein synthesis is about 6 peptide bonds per minute, thus it takes about 1 to 2 minutes to synthesize an average sized protein. Because mRNA is often several thousand nucleotides in length, the same mRNA molecules can be simultaneously bound by many ribosomes. An mRNA that is bound by multiple ribosomes is called a polysome. Polysomes provide a mechanism for many copies of a protein to be translated from a single mRNA. Polysomes in the cytosol synthesize most of the proteins and enzymes required by the body for intracellular processes such as metabolism.
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The best-studied route of uptake from the plasma membrane is via the clathrin-coated pit. Clathrin assembles into a lattice structure that deforms the membrane into a bud (). The main structural units of the clathrin cage are the heavy chain (CHC-1) and the light chain (CLIC-1). Extensive evidence, from RNAi experiments and a temperature sensitive allele of , indicates that CHC-1 is essential for endocytosis and viability in (). CLIC-1 itself does not appear to be essential for endocytosis or viability, based upon RNAi and deletion mutant analysis (). However, RNAi of in a temperature sensitive mutant background caused embryonic lethality at the permissive temperature, suggesting a role of CLIC-1 in at least one clathrin-mediated function (). Data in mammalian cells indicates that clathrin light chain is more important for clathrin-mediated transport from the Golgi to endosomes, a route that delivers newly synthesized lysosomal proteins, but this has not been tested in worms ().
After it is synthesized disulfide bonds are formed and the protein folds into its three dimensional state. Some proteins require post-translational modification before becoming fully active. These modifications can include removal of segments using peptidases, addition of phosphate, sugar or lipids to specific amino acids and glycosylation.
A number of converging lines of research suggest that RME-1 and its mammalian homologs are membrane fission molecules that act on endosomal membranes in a manner similar to the well-studied mechanoenzyme dynamin. The large GTPase dynamin polymerizes to form spirals around the neck of clathrin-coated pits on the plasma membrane (). Current models indicate that near simultaneous hydrolysis of GTP by the dynamin subunits within the spiral causes a dramatic change in the pitch of the spiral, constricting the neck of the pit, strongly promoting fission and release of a free vesicle (). Thus dynamin is often called a “pinchase”. In addition, dynamin associates with other fission promoting molecules including BAR domain proteins that polymerize and tubulate membranes, and proteins that stimulate local actin polymerization (). RME-1 and its mammalian homologs are ATPases, not GTPases, but the crystal structure of one of the mammalian RME-1 homologs, EHD2, showed striking structural similarities in the ATPase domain to the GTPase domain of dynamin (; ). This similarity is significant because the GTPase domain of dynamin is the domain that polymerizes into spirals around membranes. Consistent with this idea, it was found that purified recombinant RME-1 incubated with negatively charged liposomes and ATPγS produced dynamin-like spirals around the membranes () (). RME-1 binding to membranes improved as the level of negative charged lipid levels increased. A combination of phosphatidylserine (PS) and PI(4,5)P2 produced the best binding of RME-1 to liposomes. RME-1 bound less well to PS and PI(4)P. Mammalian recycling endosomes are reported to be enriched in PI(4)P and PI(4,5)P2 (). In the intestine reporters indicate that PI(4,5)P2 is enriched on RME-1-positive basolateral recycling endosomes ().