Transmembrane domain analysis of polycystin-1, the product of the polycystic kidney disease-1 (PKD1) gene: evidence for 11 membrane-spanning domains.
Polycystin-1, the protein product of the polycystic kidney disease-1 (PKD1) gene, was originally predicted to be an integral membrane glycoprotein with 11 transmembrane (TM) domains (TM 1-11). Subsequent comparative sequence analyses led to a revision of the original model, which retained the overall topology and 11 TM segments (TM I-XI) but dropped 3 of the original domains and introduced 3 new TM domains. The membrane-spanning potential and the orientation of each of the proposed TM domains following the extracellular REJ domain (TM I-XI and TM 11) have now been tested. Using a series of N-terminal polycystin TM-glycosylation reporter gene fusions expressed in vivo, we assayed N-linked glycosylation of the C-terminal glycosylation reporter as an indicator of TM domain presence and orientation. This approach has clearly demonstrated that 7 of the 12 TM domains tested function as membrane-spanning domains. In vitro analysis of the topogenic potential of the five remaining TM domains revealed that four of these also function as membrane-spanning domains, thus supporting an 11 TM structure for polycystin-1 comprised of TM domains I-XI. In addition, these studies suggest that the membrane insertion of TM domains I-IX occurs in a cotranslational and sequential manner, while multiple topogenic determinants appear to be required for the integration of the C-terminal-most TM segments of polycystin-1.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
Nancy
N
Nims
Dianne
D
Vassmer
Robin L
RL
Maser
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D008958:Models, Molecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D050396:TRPP Cation Channels; D014162:Transfection
D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels