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 N ␣ -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 N ␣ -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 N ␣ -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. ⌬nat1 or ⌬ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while ⌬nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by ⌬nat1 or ⌬ard1 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.

The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p͞Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperonelike effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the ⌬ssz1 strain, whereas overexpression of Ssz1p did not affect the ⌬zuo1 strain, suggesting a pivotal function for the DnaJ homolog. P roteins synthesized on cytosolic ribosomes either fold or translocate to their final location cotranslationally or posttranslationally. Translocation and folding require chaperonelike proteins that often serve multiple and overlapping functions. The complex chaperone network in the eukaryotic cell is currently only poorly understood (1-3).Most posttranslational translocation events require the translocating proteins to be unfolded. This requirement is at least partly ensured by binding of cytosolic chaperones to newly translated proteins. The yeast DnaK͞Hsp70 homolog Ssa1͞2p and its partner protein, the DnaJ homolog Ydj1p, are involved in protein translocation into a variety of compartments (4). Besides their role in translocation, Ssa1͞2p and Ydj1p are involved in cytosolic protein folding, most likely in a posttranslational manner (5-7). Other chaperones assisting posttranslational folding in the eukaryotic cytosol are the chaperonin CCT and Hsp90 (2,8,9).Some chaperones interact cotranslationally with polypeptides. In both eukaryotes and prokaryotes, soluble DnaK and DnaJ homologs bind to nascent chains and subsequently assist their folding (2, 10, 11). The ribosome-bound DnaK homolog Ssb1͞2p can be crosslinked to nascent chains, providing evidence for a functionally important interaction (12).There is long-standing circumstantial evidence of cotranslational import into mitochondria (13). More recent data suggest that some precursor proteins even require a cotranslational mechanism to be imported into mitochondria (14-16). However, no specialized component of a mitochondrial cotranslational translocation system, comparable to a signal recognition particle or signal recognition particle receptor, has been identified (17).In a previous study we introduced an in vitro mitochondrial protein import assay for the identification of cytosolic components involved in either cotranslational translocation or interacting with nascent precursor proteins in a chaperone-like manner. The assay, b...

The chaperones RAC (ribosome-associated complex), consisting of Ssz1p and zuotin, and Ssb1͞2p are associated with ribosomes of yeast. Ssb1͞2p was previously shown to form a crosslink product to polypeptides trapped in ribosome-nascent chain complexes (RNCs) in vitro. Here we show that an efficient crosslink of the nascent chain to Ssb1͞2p depends on the presence of functional RAC. The crosslink to Ssb1͞2p was significantly diminished if (i) RAC was removed from RNCs: a process reversed by addition of purified RAC; (ii) RAC carried a mutation in the J-domain of zuotin, leading to its inactivation in vivo; (iii) RAC's Ssz1p subunit was absent because RNCs were generated in a ⌬ssz1-derived translation extract. In vivo the same specific set of growth defects caused by the absence of any of the three chaperones was also displayed by a ⌬ssb1͞2⌬ssz1⌬zuo1 strain. The combination of in vitro and in vivo data supports a model in which Ssb1͞2p, Ssz1p, and zuotin act in concert on nascent chains while they are being synthesized.

Cytokinesis requires the spatio-temporal coordination of cell-cycle control and cytoskeletal reorganization. Members of the Rho-family of GTPases are crucial regulators of this process and assembly of the contractile ring depends on local activation of Rho signalling. Here, we show that the armadillo protein p0071, unlike its relative p120(ctn), is localized at the midbody during cytokinesis and is essential for cell division. Both knockdown and overexpression of p0071 interfered with normal cell growth and survival due to cytokinesis defects with formation of multinucleated cells and induction of apoptosis. This failure of cytokinesis seemingly correlated with the deregulation of Rho activity in response to altered p0071 expression. The function of p0071 in regulating Rho activity occurred through an association of p0071 with RhoA, as well as the physical and functional interaction of p0071 with Ect2, the one Rho guanine-nucleotide exchange factor (GEF) essential for cytokinesis. These findings support an essential role for p0071 in spatially regulating restricted Rho signalling during cytokinesis.

SummaryDownregulation of adherens junction proteins is a frequent event in carcinogenesis. How desmosomal proteins contribute to tumor formation by regulating the balance between adhesion and proliferation is not well understood. The desmosomal protein plakophilin 1 can increase intercellular adhesion by recruiting desmosomal proteins to the plasma membrane or stimulate proliferation by enhancing translation rates. Here, we show that these dual functions of plakophilin 1 are regulated by growth factor signaling. Insulin stimulation induced the phosphorylation of plakophilin 1, which correlated with reduced intercellular adhesion and an increased activity of plakophilin 1 in the stimulation of translation. Phosphorylation was mediated by Akt2 at four motifs within the plakophilin 1 N-terminal domain. A plakophilin 1 phospho-mimetic mutant revealed reduced intercellular adhesion and accumulated in the cytoplasm, where it increased translation and proliferation rates and conferred the capacity of anchorage-independent growth. The cytoplasmic accumulation was mediated by the stabilization of phosphorylated plakophilin 1, which displayed a considerably increased half-life, whereas nonphosphorylated plakophilin 1 was more rapidly degraded. Our data indicate that upon activation of growth factor signaling, plakophilin 1 switches from a desmosome-associated growth-inhibiting to a cytoplasmic proliferation-promoting function. This supports the view that the deregulation of plakophilin 1, as observed in several tumors, directly contributes to hyperproliferation and carcinogenesis in a context-dependent manner.