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Description
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Ubiquitin-like proteins fall into two classes: the first class, ubiquitin-like modifiers (UBLs)function as modifiers in a manner analogous to that of ubiquitin.Examples of UBLs are SUMO, Rub1 (also called Nedd8), Apg8and Apg12. Proteins of the second class include parkin,RAD23 and DSK2, are designated ubiquitin-domain proteins(UDPs). These proteins contain domains that are related toubiquitin but are otherwise unrelated to each other. In contrastto UBLs, UDPs are not conjugated to other proteins. Apg8 isrequired for autophagy (intracellular bulk protein degradation)in yeast. Starved yeast cells take up their own cytoplasm intovacuoles through autophagic bodies. Autophagic bodies forma double-membraned structure called the autophagosome,which subsequently fuses with the vacuole/lysosome. Thisprocess similar in mammals. Two sets of genes, APG andAUT, have been identified with this process, and areresponsible for two ubiquitin-like systems Apg12 and Apg8,respectively. Apg12 is synthesized in its mature form andseems to have one target, Apg5. Almost all Apg12 moleculesare conjugated with Apg5. Aut2/Apg4 processes theApg8/Aut7 system at its carboxy-terminal region. Apg8 existsin two forms, one is membrane bound through a phospholipid.Lipidation/ activation of Apg8 is mediated by Apg7 andtransferred to Apg3 and finally forms a conjugate withphosphatidyl-ethanolamine (PE). Apg4 cleaves Apg8toPE,releasing Apg8 from membrane. Morphological studies showthat Apg8 localizes on the membrane of intermediate structuresof the autophagosome, this transient association seems to beessential for formation of the autophagosome. Both Apg12 andApg8 are highly conserved, with apparent homologues in theworm, mammals and plants. In higher eukaryotes, Apg8consists of a multigene family. Figure 1. Immunoblot of APG8 fusion protein. Anti-APG8 antibody generated by immunization with recombinant yeast APG8 was testedby immunoblot with other anti-UBL antibodies against E.coli lysates expressing the APG8-GFP fusion protein. All UBLs possess limitedhomology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel Ashows total protein staining using ponceau. Panel B shows specific reaction with APG8 using a 1:4,000 and 1:8,000 dilution of Antibodies-Online'sIgG fraction of Rabbit-anti-APG8 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG MX (code ABIN102010).All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4o C.E.coli lysate proteins were separated by SDS-PAGE using a 15% gel. Similar experiments (data not shown), where other UBL fusion proteinswere separated and probed with this antibody showed no reactivity of anti-APG8 with other UBLs. This data indicates that anti-APG8 is highlyspecific and does not cross react with other UBLs. A chemiluminescence system was used for signal detection (Roche). Other detectionsystems will yield similar results. Data contributed by M. Malakhov, www.lifesensors.com , personal communication. a n ti-Rub 1 } } } } } } Apg 8-GFP Apg 8-GFP 1: 1000 1: 2000 1: 1000 1: 500 1: 2000 1: 1000 1: 1000 1: 2000 1: 2000 1: 4000 1: 8000 1: 1000 1: 4000 } A B an ti -S U M O an ti -U b a n ti-H u b 1 an ti -U rm 1 an ti-Ap g 8 an ti-A p g 12 1:1000 A B
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