Unique D box and KEN box sequences limit ubiquitination of Acm1 and promote pseudosubstrate inhibition of the anaphase-promoting complex.

The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC(Cdh1)) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC(Cdh1) inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC(Cdh1) inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC(Cdh1) in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation.

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification.

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.

DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair.

The yeast Mlh1-Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1-Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.

Effects of TCDD upon IkappaB and IKK subunits localized in microsomes by proteomics.

Biochemical studies have shown that microsomes represent an important subcellular fraction for determining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects. Proteomic analysis by two-dimensional gel-mass spectrometry of liver microsomes was undertaken to gain new insight into the actions of TCDD in male and female rats. Proteomic analysis showed TCDD induced several xenobiotic metabolism enzymes as well as a protein at 90kDa identified by mass spectrometry as IkappaB kinase beta/IKK2. This observation led to the discovery of other NF-kappaB binding proteins and kinases in microsomes and effects by TCDD. Western blotting for IKK and IkappaB family members in microsomes showed a distinct pattern from cytosol. IKK1 and IKK2 were both present in microsomes and were catalytically active although, unlike cytosol, IKKgamma/NEMO was not detectable. TCDD exposure produced an elevation in cytosolic and microsomal IKK activity of both genders. The NF-kappaB binding proteins IkappaBbeta and IkappaBgamma were prevalent in microsomes, while IkappaBalpha and IkappaB epsilon proteins were absent. TCDD treatment produced hyperphosphorylation of microsomal IkappaBbeta in both sexes with females being most sensitive. In cytosol, IkappaBalpha, IkappaBbeta, and IkappaB epsilon, but not IkappaBgamma, were clearly observed but were not changed by TCDD. Overall, proteomic analysis indicated the presence of NF-kappaB pathway members in microsomes, selectively altered by dioxin, which may influence immune and inflammatory responses within the liver.