Browsing by Author "Iyappan, Saravanakumar"

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The function of the beta6/Pre7 propeptide for 20S proteasome biogenesis in baker’s yeast
(2004) Iyappan, Saravanakumar; Heinemeyer, Wolfgang (PD Dr. )
In eukaryotes, the regulated proteolysis of intracellular proteins occurs through a specialized enzymatic machinery, the ubiquitin-proteasome system. Here proteins are specifically recognized and marked for degradation by addition of poly-ubiquitin chains before being degraded by the 26S proteasome. The 20S proteasome is the catalytic core of the 26S proteasome, composed of two copies of each 7 different alpha- and beta-type subunits forming a barrel-shaped complex. Assembly and maturation of the eukaryotic 20S proteasome is a multi-step process, in which the free alpha- and beta-type subunits are first assembled into the complete but still inactive complex. Final activation occurs after conversion of three of the beta-type subunits into their mature form by autocatalytic removal of N-terminal propeptides leading to the exposure of catalytic threonines at their N-termini. The contribution of the propeptides of these active beta-type subunits to particle assembly in baker's yeast ranges from absolute necessity to dispensability. In addition, of the four inactive beta-type subunits three are also synthesized with a propeptide which stays either unprocessed (beta-3/Pup3) or is only partially removed (beta-7/Pre4 and beta-6/Pre7) by the action of the activated neighbour beta-type subunits. The resulting short N-terminal extensions of 8-9 residues are found in the crystal structure of the yeast 20S proteasome in an extended conformation each reaching the restrictions separating the central proteolytic chamber from the antechambers. In this work multiple questions are addressed regarding the function of propeptides of the inactive subunits that have been preserved during proteasome evolution. The work focusses especially on the structure and function of the beta-6/Pre7 propeptide and its contribution to proteasome biogenesis, because it is essential for yeast cell viability in contrast to the beta-3/Pup3 propeptide that is dispensable for proteasome function, like already reported for the beta7/Pre4 propeptide. The region in the beta-6/Pre7 propeptide that is cleaved off by the neighboring active beta-type subunit beta-2/Pup1 is not essential, but the remaining 9 residues found in the mature proteasome are indispensable for its function. Stepwise truncation analysis in the Pre7 propeptide remnant region revealed that the six most C-terminal amino acids are sufficient for cell survival, but cell growth is considerably retarded if this piece is shortened from 9 to 6 residues. Surprisingly, the need for this propeptide remnant is restricted to the presence of the proteasome assembly factor Ump1. Ump1 was shown to enhance the dimerization of two half-proteasome precursor complexes and in addition to promote proteasome maturation. Fractionation of precursor and mature proteasome species by gel-filtration analysis as well as pulse-chase analysis elucidated that in a UMP1 wild-type strain background the Pre7 mutant bearing a propeptide remnant with 6 residues has severe defects in particle assembly and maturation. In contrast to this, in ump deletion cells such perturbation in assembly and maturation was found even in the presence of wild-type Pre7 and these defects were not enhanced by the truncations in the Pre7 propeptide region. Similarly, the proteasomal peptidase activities were reduced gradually along with stepwise truncations in the Pre7 propeptide region in the presence of Ump1, but the activity profiles were not altered in the ump1deletion strain background. As seen in non-denaturing gel electrophoresis, truncations in the Pre7 propeptide remnant region by up to 3 residues interfered with the dimerization of two half-proteasomes to the pre-holoproteasome and thus led to accumulation of these assembly products, but again only in the presence of Ump1. The obstruction stage in the proteasome assembly in those strains that are viable only in the absence of Ump1 was analysed by expressing the Ump1 protein from an inducible promoter. As expected, in cells carrying a Pre7 propeptide remnant shorter than 6 residues, the appearance of Ump1 completely inhibited their proliferation after few cell divisions. Analysis of these cells revealed that the proteasome assembly process is blocked at the state of half-proteasome assembly intermediates containing Ump1. Furthermore, interaction of Ump1 was restricted to the half-proteasome, whereas in strains carrying wild-type Pre7 Ump1 was found in a putative earlier assembly intermediate. Thus, the binding of Ump1 to early assembly stages might trigger the dimerization process more efficiently than its interaction with completely assembled half-proteasomes. In vitro interaction and GST-pull down analysis additionally indicated that formation of half-proteasomes is coupled with incorporation of Ump1 and that its association depends on the late incorporating subunits Pre2, Pre4 and Pre7, most likely mediated by their propeptides. Replacement analysis of the Pre7 propeptide remnant region showed that its function specifically depends on its amino acid sequence. Sequence comparison of the primary structure of beta-6 orthologues from different species enlightened that a proline at position -6 and a tyrosine at -5 have been absolutely conserved during evolution. In addition to this, a stretch of hydrophobic amino acids that forms a hydrophobic pocket and binds the propeptide in the mature proteasome was also found to be conserved in all known beta 6 subunits. The tyrosine at position -5 turned out to be crucial for the function of the Pre7 propeptide remnant, since its exchange with alanine was lethal in UMP1 wild-type cells. The aromatic side chain of this tyrosine is required to fix the propeptide remnant on the Pre7 subunit surface by interacting with the hydrophobic pocket, whereas the hydroxyl group is dispensable. Two phenylalanines in the hydrophobic pocket were also found to be essential, suggesting that these residues are either crucial to fix the propeptide at any stage of the assembly process or required for the structural integrity of the Pre7 subunit. Remarkably, the lethality caused by the mutations in the hydrophobic pocket was again coupled to the presence of the Ump1 protein. Probably, the exposure of this hydrophobic surface due to the absence of the Pre7 propeptide or due to loss of its ability to bind it might lead to unfavorable interactions of the Ump1 protein with this area and so might preclude further assembly and maturation steps. Although the propeptide of the Pre7 subunit seems to be crucial to avoid such unfavorable interactions of Ump1 with the hydrophobic surface, a primary role might lie in maintaining a structural integrity, which is a prerequisite for the proceeding of the assembly and maturation process. This is implicated by the fact that replacing the tyrosine in the propeptide region in combination with replacements of any of the hydrophobic amino acids in the hydrophobic pocket causes severe growth defects even in ump1 deletion cells and that exchanging all conserved residues in the pocket by alanines is lethal. Taken together this work establishes a critical role of Pre7 propeptide in the yeast 20S proteasome assembly and in its maturation. The conserved residues found in the propeptide and in the hydrophobic pocket of the Pre7 subunit are concertedly necessary for proper folding into a tertiary structure that allows efficient incorporation of the subunit into the proteasome. Moreover, the propeptide of Pre7 is specifically required for the function of the proteasome assembly and maturation factor Ump1 during proteasome biogenesis. In addition, it might be involved in interactions with other proteasomal proteins or in the transmission of structural alterations in the proteasomal assembly intermediates.