Secretion of recombinant pro- and mature fungal alpha-sarcin ribotoxin by the methylotrophic yeast Pichia pastoris: the Lys-Arg motif is required for maturation.
alpha-Sarcin is a ribosome-inactivating protein from the mold Aspergillus giganteus. The methylotrophic yeast Pichia pastoris has been transformed with two plasmids (pHILD2prealphaS and pHILS1prealphaS), which contain the complete alpha-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence of P. pastoris acid phosphatase. The transformed yeasts secreted both mature and pro-alpha-sarcin. The presence of this pro-alpha-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the alpha-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant alpha-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-alpha-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.
Martínez-Ruiz
A
A
Facultad de Química, Universidad Complutense, Madrid, E-28040, Spain.
Martínez del Pozo
A
A
Lacadena
J
J
Mancheño
J M
JM
Oñaderra
M
M
López-otín
C
C
Gavilanes
J G
JG
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003603:Cytotoxins; D004271:DNA, Fungal; D005656:Fungal Proteins; D009183:Mycotoxins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C014801:alpha-sarcin; D004722:Endoribonucleases