Membrane Association and Catabolite Repression of the Sulfolobus solfataricus α-Amylase.
Sulfolobus solfataricus is a thermoacidophilic member of the archaea whose envelope consists of an ether-linked lipid monolayer surrounded by a protein S-layer. Protein translocation across this envelope must accommodate a steep proton gradient that is subject to temperature extremes. To better understand this process in vivo, studies were conducted on the S. solfataricus glycosyl hydrolyase family 57 α-Amylase (AmyA). Cell lines harboring site specific modifications of the amyA promoter and AmyA structural domains were created by gene replacement using markerless exchange and characterized by Western blot, enzyme assay and culture-based analysis. Fusion of amyA to the malAp promoter overcame amyAp-mediated regulatory responses to media composition including glucose and amino acid repression implicating action act at the level of transcription. Deletion of the AmyA Class II N-terminal signal peptide blocked protein secretion and intracellular protein accumulation. Deletion analysis of a conserved bipartite C-terminal motif consisting of a hydrophobic region followed by several charged residues indicated the charged residues played an essential role in membrane-association but not protein secretion. Mutants lacking the C-terminal bipartite motif exhibited reduced growth rates on starch as the sole carbon and energy source; therefore, association of AmyA with the membrane improves carbohydrate utilization. Widespread occurrence of this motif in other secreted proteins of S. solfataricus and of related Crenarchaeota suggests protein association with membranes is a general trait used by these organisms to influence external processes.
School of Biological Sciences, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln 68588, NE, USA. edith_soo@yahoo.com.
Edith
E
Soo
School of Biological Sciences, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln 68588, NE, USA. deepakgr@unl.edu.
Deepak
D
Rudrappa
School of Biological Sciences, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln 68588, NE, USA. pblum1@unl.edu.
Paul
P
Blum
Sulfolobus; archaea; catabolite repression; secretion; α-amylase
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