Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway.

Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to "iron-responsive elements" within their untranslated regions. In iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster (ISC) that inhibits RNA-binding activity and converts the protein to cytosolic aconitase. We show that the constitutive IRP1(C437S) mutant, which fails to form an ISC, is destabilized by iron. Thus, exposure of H1299 cells to ferric ammonium citrate reduced the half-life of transfected IRP1(C437S) from approximately 24 h to approximately 10 h. The iron-dependent degradation of IRP1(C437S) involved ubiquitination, required ongoing transcription and translation, and could be efficiently blocked by the proteasomal inhibitors MG132 and lactacystin. Similar results were obtained with overexpressed wild-type IRP1, which predominated in the apo-form even in iron-loaded H1299 cells, possibly due to saturation of the ISC assembly machinery. Importantly, inhibition of ISC biogenesis in HeLa cells by small interfering RNA knockdown of the cysteine desulfurase Nfs1 sensitized endogenous IRP1 for iron-dependent degradation. Collectively, these data uncover a mechanism for the regulation of IRP1 abundance as a means to control its RNA-binding activity, when the ISC assembly pathway is impaired.

Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a complex process involving more than 20 components. So far, functional investigations have mainly been performed in Saccharomyces cerevisiae. Here, we have analyzed the role of the human cysteine desulfurase Nfs1 (huNfs1), which serves as a sulfur donor in biogenesis. The protein is located predominantly in mitochondria, but small amounts are present in the cytosol/nucleus. huNfs1 was depleted efficiently in HeLa cells by a small interfering RNA (siRNA) approach, resulting in a drastic growth retardation and striking morphological changes of mitochondria. The activities of both mitochondrial and cytosolic Fe/S proteins were strongly impaired, demonstrating that huNfs1 performs an essential function in Fe/S protein biogenesis in human cells. Expression of murine Nfs1 (muNfs1) in huNfs1-depleted cells restored both growth and Fe/S protein activities to wild-type levels, indicating the specificity of the siRNA depletion approach. No complementation of the growth retardation was observed, when muNfs1 was synthesized without its mitochondrial presequence. This extramitochondrial muNfs1 did not support maintenance of Fe/S protein activities, neither in the cytosol nor in mitochondria. In conclusion, our study shows that the essential huNfs1 is required inside mitochondria for efficient maturation of cellular Fe/S proteins. The results have implications for the regulation of iron homeostasis by cytosolic iron regulatory protein 1.

Investigation of iron-sulfur protein maturation in eukaryotes.

Iron-sulfur (Fe-S) clusters are cofactors of many proteins that are involved in central biochemical pathways, such as oxidative phosphorylation, photosynthesis, and amino acid biosynthesis. The assembly of these cofactors and the maturation of Fe-S proteins require complex cellular machineries in all kingdoms of life. In eukaryotes, Fe-S protein biogenesis is an essential process, and mitochondria perform a primary role in synthesis. Defects in Fe-S protein maturation in yeast result in respiratory deficiency and auxotrophies for certain amino acids and vitamins that require Fe-S proteins for their biosynthesis. Frequently, heme biosynthesis is also affected. The present compendium describes assays for the analysis of de novo Fe-S cluster and heme formation, cellular iron homeostasis, and the activity of Fe-S cluster- and heme-containing enzymes. These approaches are crucial to elucidate the mechanisms underlying the maturation of Fe-S proteins and may aid in the identification of new members of this evolutionary ancient process.

Serglycin proteoglycan is sorted into zymogen granules of rat pancreatic acinar cells.

Serglycin is known as a secretory granule proteoglyean in hematopoietic cells. In this study we identified a high-molecular-weight molecule in aggregated content proteins of zymogen granules of pancreatic acinar cells. The amino acid composition of the isolated protein showed high similarity to serglycin proteoglycan core protein. To confirm the expression of serglycin proteoglycan in pancreatic acinar cells we cloned the rat pancreas cDNA of serglycin core protein and detected the serglycin mRNA in pancreas tissue and AR4-2J cells by reverse transcription-PCR. In AR4-2J cells, transfected with serglycin fused to green fluorescent protein (EGFP), serglycin localized within a web-like pattern in the perinuclear space as well as with a punctate pattern distributed in the cytoplasm. The perinuclear structures colocalized with the Golgi membrane-associated protein p115 and the punctate structures with the secretory enzyme procarboxypeptidase A, indicating that the serglycin-EGFP fusion protein travels through compartments of the secretory pathway and is sorted into secretory granules. Using an antiserum against serglycin core protein immunofluorescence as well as immunogold electron microscopy analysis conrirmed the subcellular distribution of serglycin proteoglycan in zymogen granules of pancreatic acinar cells. To prevent glycosylation of serglycin core protein we incubated AR4-2J cells with 2 mM p-nitrophenyl-beta-D-xylopyranoside (PNP-xyloside), which serves as alternate substrate for glycosaminoglycan chain attachment. Furthermore, we deleted the serine/glycine repeat region in the serglycin core protein. In both approaches the transfected serglycin-EGFP fusion protein could be detected predominantly in perinuclear Golgi membrane structures, while in control cells the serglycin fusion protein was mostly sorted into the secretory granules. Additionally, we show that sorting of secretory enzymes like amylase

The VSFASSQQ motif confers calcium sensitivity to the intracellular apyrase LALP70.

BACKGROUND: Apyrases are divalent ion dependent tri- and dinucleotide phosphatases with different substrate specificity. The intracellular lysosomal apyrase LALP70 is also expressed as a splice variant (LALP70v) lacking a VSFASSQQ motif in the center of the molecule (aminoacids 287-294). However, the functional significance of this motif is unknown. In this report we used a thin layer chromatography approach to study separately the UTPase and UDPase activity of the two LALP-enzymes. RESULTS: We show, that LALP70 and LALP70v cleaved UTP to UDP in a calcium independent manner. In contrast, the cleavage of UDP to UMP was strongly calcium dependent for LALP70, but calcium independent for LALP70v. CONCLUSIONS: The VSFASSQQ motif not only influences the substrate specificity of LALP70, but it confers calcium sensitivity to LALP70 during the UDP cleavage. Whether this is due to direct binding of calcium to this motif or to a conformational change of the enzyme, remains to be elucidated.