Research Report 2010 - MDC

Figure 2. Processing of N-linked glycans in the yeast and mammalian ER. (a) A glucose 3 -mannose9-N-acetylglucosamine 2 oligosaccharide is initiallyattached to asparagine in NXS/T motifs by the oliglsyccharyltransferase. Gls1 (glucosidase 1) and Gls2 remove the two outmost glucoseresidues and generate a glucose 1 -mannose 9 -N-acetylglucosamine 2 sugar (step 1). Further processing by Gls2 results in a mannose 9 -N-acetylglucosamine2 glycan that protects the glycoprotein from disposal (step 2). Mns1 trims the outmost mannose moiety of the B branch, yielding a mannose8 -N-actelyglucosamine 2 oligosaccharide, which indicates a protein that is retained in the ER for a prolonged period of time (step 3).Subsequently Htm1 processes the C-branch yielding a mannose7-N-actelyglucosamine2 oligosaccharide (step 4). (b) In contrast to yeast, in mammaliancells removal of the residual glucose residue from the A branch by glucosidase-II (GII, step 2) is reversible. GT reglucosylates glycoproteinsthat have not attained their native fold (step 3). Extensive demannosylation by ERManI and probably the EDEM proteins yield a mannose 5 -Nacetylglucosamine2 glycan that is a signal for disposal (step 4). Light blue mannose residues protect the glycoprotein from degradation until theyare removed.mic 26S-proteasomes. Specific functions, like substrateselection and ubiquitylation, are assigned to distinctsubunits of the HRD-ligase. For example substratebinding involves the ER-luminal domain of Hrd3 andthe associated lectin Yos9. Ubiquitylation depends onthe RING-finger protein Hrd1 and the ubiquitin-conjugatingenzyme Ubc7 and its co-factor Cue1. The drivingforce for dislocation is provided by ATP hydrolysis at thehexameric Cdc48 (in mammals p97 or VCP) complex. Itconsists of Cdc48 itself and the associated co-factorsUfd1 and Npl4. This ATPase binds to Ubx2 and in additionto poly-ubiquitylated substrates at the ligases. Twoadditional subunits of the ligase complex have beenidentified, Der1 and Usa1, but their precise function isstill under debate. It is furthermore speculated that theligase forms a channel in the membrane through whichsubstrates are dislocated. However, other channels havebeen discussed as well and direct evidence for thechannel hypothesis is still missing.By biochemical analyses we were now able to showthat Usa1 plays a dual role within the HRD ligase complex:First, it recruits the ancillary factor Der1 and secondit mediates oligomerization of the HRD ubiquitinligase.These separate activities mirror different requirementsfor the processing of malfolded proteins withdistinct topology. On one hand, Usa1 promotedoligomerization of Hrd1 is predominantly required forthe breakdown of membrane proteins while it is dispensablefor the turnover of soluble polypeptides. Onthe other hand, Der1 recruitment, in turn, is a prerequisiteonly for the degradation of soluble substrates. Inthis collaborative work with the group of Udo104 Cancer Research