Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S.
lambda S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including lambda S and 21 S, have the "dual-start" regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic "helical-hairpin" topology, with N and C termini on the cytoplasmic side of the membrane. However, lambda S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of lambda S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin S(lambda) has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.
Gründling
A
A
Department of Biochemistry and Biophysics Texas A&M University, College Station, Texas 77843-2128, USA.
Bläsi
U
U
Young
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R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D003062:Codon; D003545:Cysteine; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C108624:S holin, bacteriophage lambda; D014764:Viral Proteins; D003545:Cysteine