C Yeang et al/ Biochimica et Biophysica Acta 1781(2008 )610-617 SMS1 SMS2 國回國画包 MS1ASAM 國國國國 SAM-SMS2 國國國國國 NBD-SM NBD-SM 50 KD 40 kD a Mannosidase‖l Flag TOPRO-3 Merge Cadherin Flag TOPRO-3 Merge Fig. 5. Analysis of potentially important domains for the differential localization of SMSI and SMS2. (A)Schematic of the Flag-tagged constructs used. SAM: Sterile Alpha Motif. TM transmembrane domain. (B)SMS1ASAM-Flag, a mutant with truncation of the first 61 amino acids from SMSl, shows no defect in SMs activity compared with WT SMSl-Flag. Likewise, SAM-SMS2, a fusion protein comprised of the SAM domain from SMSI(amino acids 1-61)and the entirety of sMS2, has unaltered sMs activity compared to wT-SMS2-Flag depicted in red. TOPR ch enzyme is shown at the bottom.(C) SMS1ASAM-Flag remains as a Golgi targeted protein. a Mannosidase ll is depicted in green, while SMSIASAM-Flag is depicted in red. TOPRO-3 is in blue of SAM from SMSI or addition of SAM to SMS2 has no specific properties for both SMSI and SMSZ. This aspect deserves ble impact on SMS activity. Since SMs activity is on. For an unknown reason SMSl-Flag western blot Figs. 2A and 5B). The up band with correct impact on the process of disease development. to of SMSI-Flag disappeared in truncated SMSl-Flag mains could provide a molecular basis for studying isoform- transfected cells( Fig 5B).deletion of SAM from SMS1 or addition of SAM to SMS2 has no appreciable impact on SMS activity. Since SMS activity is potentially important in certain diseases, and SMS1 and SMS2 activity may have a different impact on the process of disease development, to elucidate these domains could provide a molecular basis for studying isoform￾specific properties for both SMS1 and SMS2. This aspect deserves further investigation. For an unknown reason SMS1-Flag western blot showed a doublet (Figs. 2A and 5B). The up band with correct molecular weight of SMS1-Flag disappeared in truncated SMS1-Flag transfected cells (Fig. 5B). Fig. 5. Analysis of potentially important domains for the differential localization of SMS1 and SMS2. (A) Schematic of the Flag-tagged constructs used. SAM: Sterile Alpha Motif. TM: transmembrane domain. (B) SMS1ΔSAM-Flag, a mutant with truncation of the first 61 amino acids from SMS1, shows no defect in SMS activity compared with WT SMS1-Flag. Likewise, SAM-SMS2, a fusion protein comprised of the SAM domain from SMS1 (amino acids 1–61) and the entirety of SMS2, has unaltered SMS activity compared to WT-SMS2-Flag. The protein levels of each enzyme is shown at the bottom. (C) SMS1ΔSAM-Flag remains as a Golgi targeted protein. α Mannosidase II is depicted in green, while SMS1ΔSAM-Flag is depicted in red. TOPRO-3 is in blue. (D) SAM-SMS2-Flag exhibits an identical localization pattern compared to wild type SMS2. Cadherin is depicted in green, while SAM–SMS2-Flag is depicted in red. TOPRO-3 is in blue. 616 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617