Characterization of an insect ferritin subunit synthesized in a cell-free system.
Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28-30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28-30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.
Ketola-Pirie
C A
CA
Department of Zoology, University of Western Ontario, London, Canada.
D000818:Animals; D002474:Cell-Free System; D003208:Concanavalin A / Q000378:metabolism; D019286:Endopeptidase K; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry*; D006031:Glycosylation / Q000187:drug effects; D007501:Iron / Q000502:physiology; D007814:Larva; D007915:Lepidoptera; D008970:Molecular Weight; D011061:Poly A; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012697:Serine Endopeptidases; D014415:Tunicamycin / Q000494:pharmacology
D012333:RNA, Messenger; D003208:Concanavalin A; D014415:Tunicamycin; D011061:Poly A; D005293:Ferritins; D007501:Iron; D012697:Serine Endopeptidases; D019286:Endopeptidase K