The ubiquitin-proteasome proteolytic pathway is pivotal in most biological processes. Despite a great level of information available for the eukaryotic 26S proteasome-the protease responsible for the degradation of ubiquitylated proteinsseveral structural and functional questions remain unanswered. To gain more insight into the assembly and function of the metazoan 26S proteasome, a two-hybrid-based protein interaction map was generated using 30 Caenorhabditis elegans proteasome subunits. The results recapitulate interactions reported for other organisms and reveal new potential interactions both within the 19S regulatory complex and between the 19S and 20S subcomplexes. Moreover, novel potential proteasome interactors were identified, including an E3 ubiquitin ligase, transcription factors, chaperone proteins and other proteins not yet functionally annotated. By providing a wealth of novel biological hypotheses, this interaction map constitutes a framework for further analysis of the ubiquitin-proteasome pathway in a multicellular organism amenable to both classical genetics and functional genomics.

7Rational approaches to modifying cells to make molecules of interest are of 8 substantial economic and scientific interest. Most of these efforts are aimed at 9 the production of native metabolites, expression of heterologous biosynthetic 10 pathways, or protein expression. Reviews of these topics have largely focused on 11 individual strategies or cell types, but collectively they fall under the broad 12 umbrella of a growing field known as cell factory engineering. Here we condense 13 >130 reviews and key studies in the art into a metareview of cell factory 14 engineering. We identified 33 generic strategies in the field, all applicable to 15 multiple types of cells and products, and proven successful in multiple major cell 16 types. These apply to three major categories: Production of native metabolites 17 and/or bioactives, heterologous expression of biosynthetic pathways, and 18 protein expression This metareview provides general strategy guides for the 19 broad range of applications of rational engineering of cell factories. 20Introduction 21

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P 2 -P 1 -P 1 -P 2 -tyrosine(NO 2 )-aspartic acid, in which cleavage occurs between P 1 and P 1 , showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P 2 . Arginine was preferred to glutamine at P 1 , whereas proline at P 2 , P 1 , or P 1 greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K m values.Two EPs known to play a central role in the breakdown of barley (Hordeum vulgare L.) endosperm storage proteins (hordeins) are Cys EPs designated EP-A (Koehler and Ho, 1990a) and EP-B (Koehler and Ho, 1988). They are secreted by the scutellum and aleurone layer into the starchy endosperm during germination in response to GA 3 (Koehler and Ho, 1990b;Marttila et al., 1995). EP-B has an apparent molecular mass of 30 kD, is identical to MEP-1 (Phillips and Wallace, 1989), and has a possible homolog with an apparent molecular mass of 31 kD (Zhang and Jones, 1996). The substrate specificity of this EP-B has been determined from the cleavage patterns of small proteins such as hordothionin, levitide, and cholecystokinin (Poulle and Jones, 1988;Zhang and Jones, 1996). There is little information about the substrate specificity of EP-A, although both EP-A and EP-B have been shown to digest hordein (Phillips and Wallace, 1989; Ho 1990a, 1990b). Characterization of the substrate specificity of barley Cys EPs provides an essential basis for an understanding of the activation of ␤-amylase by MEP-1 (ϭ EP-B) (Guerin et al., 1992) and limit dextrinase (Sissons, 1996) during germination, in which these enzymes are released (and activated) from bound and latent forms by proteolytic cleavage.Hordeins are unusual proteins, the amino acid composition and water insolubility of which present special problems as protease substrates. Pro and Gln comprise 40% of the total amino acids, and Pro causes particular problems for EPs, especially when it is at the protease scissile bond P 1 -P 1 Ј. (The substrate positions are denoted P i , . . . , P 2 , P 1 , P 1 Ј, P 2 Ј, . . . , P j , in correspondence with the binding subsites S i , etc., according to Berger and Schechter 1970). Hordeins, which are stored as compact protein bodies within the vacuoles of endosperm cells, comprise four major clas...