The hydrophilic and acidic N-terminus of the integral membrane enzyme phosphatidylserine synthase is required for efficient membrane insertion.
The product of the yeast CHO 1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1.2 kb fragment containing the regulatory and structural components of the CHO 1 gene was sequenced. Transcription initiation in wild-type cells was found to occur between -1 and -15 relative to the first ATG of a large open reading frame capable of encoding a 30,804 molecular weight protein. This translation initiation site was active in vivo and in vitro in a heterologous system. In both cases it supported production of a protein of approximately 30,000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22,400 molecular weight. A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22,000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a cho 1 null-mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast. Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.
Sperka-Gottlieb
C
C
Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Austria.
Fasch
E V
EV
Kuchler
K
K
Bailis
A M
AM
Henry
S A
SA
Paltauf
F
F
Kohlwein
S D
SD
D000595:Amino Acid Sequence; D001483:Base Sequence; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D003600:Cytosol / Q000201:enzymology; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000378:metabolism*; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase