Yves Dubaquie

The yeast N-alpha-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides

ABSTRACT The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast Nα -acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the Nα -acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for Nα -acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Δnat1 or Δard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Δnat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Δnat1 or Δard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10

The mechanism of chaperonin‐assisted protein folding has been mostly analyzed in vitro using non‐homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15–90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre‐existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.

Hsp60-independent protein folding in the matrix of yeast mitochondria.

Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild‐type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix‐targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding.

Binding Protein-3-Selective Insulin-Like Growth Factor I Variants: Engineering, Biodistributions, and Clearance

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.

Purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10.

Publisher Summary The chapter presents a study on the purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10. The chapter describes a large-scale purification procedure of S. cerevisiae hsp60 expressed in Pichia pastoris. Yeast cpnl0 was isolated by taking advantage of the fact that bacterial GroEL forms a functional complex with co-chaperonins from mitochondria or chloroplasts. Chaperonin system is not involved in the folding of all mitochondrial proteins. Assaying the interaction of purified hsp60 and cpnl 0 with authentic substrate proteins should give insight into the specificity of the mitochondrial chaperonin system. In addition, a comparison of the bacterial GroEL/ES system with the eukaryotic hsp60/cpnl0 system may help to elucidate the chaperonin reaction cycle. The chapter describes the procedure for purification of recombinant heat shock protein 60, purification of recombinant authentic and hexahistidine-tagged chaperonin 10, and also mentions the characterization of purified yeast chaperonins.

Mapping the binding epitopes of IGF-1 and a phage-library derived peptide that inhibits IGFBP-1 binding to insulin-like growth factor

Insulin-like growth factor (IGF-1) binds to at least six different human binding proteins (BPs), which modulate its half-life and activity [1,2]. We have searched for small, structured peptides for binding to the IGF binding proteins so as to inhibit IGF binding to the BPS and thereby regulate the concentration and distribution of IGF in vivo. Indeed, variants of IGF-1 have been reported in which receptor-binding has been disrupted yet BPbinding and in vivo activity are still seen [3, 4]. Peptide or small-molecule inhibitors of the binding proteins might have similar effects, through displacement of or competition with IGF-1 for binding to BPs [4]. By displaying small, fixed-disulfide peptide libraries on bacteriophage, we selected a peptide, bp1-01, having submicromolar affinity for IGFBP-1 and having a well-defined turn-helix structure in solution [4]. To gain an understanding of how this peptide inhibits IGF-1 binding, how it might be improved in affinity, and how small-molecule mimics might be derived from it, we have further randomized and selected peptide-phage libraries and characterized synthetic peptide analogs.