Research Report 2010 - MDC

Heinemann, we could also identify the relevantdomains of Usa1, which are involved in the relevant proteininteractions at the ligase complex. The N-terminusof Usa1 binds the very C-terminal 40amino acids ofHrd1. Binding of Der1 involves the C-terminal region ofUsa1. Thus, Usa1 functions as a scaffold that assemblesthe different activities of the HRD-ligase for processingof different classes substrates that differ in their topology.In a broader sense, our data strengthen the viewthat scaffold proteins modulate ubiquitin ligase activitiesrather then being passive devices.Htm1 protein generates the N-glycan signal forglycoprotein degradation in the endoplasmicreticulumChristian Hirsch in collaboration with Simone Clerc,Daniela Maria Oggier, Paola Deprez, Claude Jakob, andMarkus AebiN-linked glycans are essential for the breakdown of glycoproteins.The covalently attached oligosaccharidestructure is used as a signal to display the folding statusof the protein. Newly synthesized proteins receive aGlc3Man9GlcNAc2 modification. Such a glycan structureprotects a newly synthesized protein from degradation.Subsequently it is trimmed by glucosidases andmannosidases until a specific signal is generated, whichis recognized by the quality control ubiquitin ligase.Since trimming of glycans is slow, these processingsteps provides a time window in which a newly synthesizedprotein can adopt its cognate conformation.In a collaborative effort we were able to define thefunction of Htm1 as an α1,2-specific exo-mannosidasethat generates the Man7GlcNAc2 oligosaccharide witha terminal α1,6-linked mannosyl residue on degradationsubstrates. This oligosaccharide signal is decodedby the ER-localized lectin Yos9 that in conjunction withHrd3 triggers the ubiquitin-proteasome dependenthydrolysis of these glycoproteins. The Htm1 exo-mannosidaseactivity requires processing of the N-glycan byglucosidase I, II and mannosidase I, resulting in asequential order of specific N-glycan structures thatreflect the folding status of the glycoprotein (Fig. 2).Since Htm1 generates the crucial signal that flags a proteinfor degradation, its activity must be tightly controlled.Thus, we searched for associated factors thatcould be involved in controlling Htm1 activity.Surprisingly, we co-purified the protein disulfide isomerasePdi1 together with Htm1. Binding of Pdi1 occursat the Htm1 C-terminus whose function is unknown.Our results raise the sepculation that the activity ofHtm1 is linked to incorrect disulfide bridge formation.Selected PublicationsNeuber, O., Jarosch,E., Volkwein, C., Walter, J., and Sommer, T. (2005)Ubx2/Sel1p links the Cdc48p/p97-Complex to Endoplasmic ReticulumAssociated Protein Degradation. Nature Cell Biol., 7, 993-998.Horn, S., Hanna, J., Hirsch, C., Volkwein, V., Schütz, A., Heinemann, U.,Sommer, T., and Jarosch, E. (2009) Usa1 functions as a scaffold of theHRD-ubiquitin ligase. Mol Cell 36, 782-793Clerc, S., Hirsch, C., Oggier, D. M., Deprez, P., Jakob, C., Sommer, T., and Aebi,M. (2009) HTM1 protein generates the N-glycan signal for glycoproteindegradation in the endoplasmic reticulum. J. Cell. Biol. 184, 159-172Hirsch, C., Gauss, R., Horn, S.C., Neuber, O., and Sommer, T. (2009) The ubiquitylationmachinery of the endoplasmic reticulum. Nature 458, 453-460.Gauss, R., Jarosch, E., Sommer, T., and Hirsch, C. (2006) A complex of Yos9pand the HRD ligase integrates endoplasmic reticulum quality control intothe degradation machinery. Nature Cell Biol., 8,849-854Cancer Research 105