Entamoeba histolytica: biochemical and molecular insights into the activities within microsomal fractions.
One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.
Salgado
Milena
M
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología (UNAM), Cuernavaca, Morelos, Mexico.
Villagómez-Castro
Julio C
JC
Rocha-Rodríguez
Rocío
R
Sabanero-López
Myrna
M
Ramos
Marco A
MA
Alagón
Alejandro
A
López-Romero
Everardo
E
Sánchez-López
Rosana
R
D000118:Acetylglucosaminidase / Q000032:analysis; D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004286:Dolichols / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000502:physiology; D004748:Entamoeba histolytica / Q000201:enzymology / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D005964:Glucosyltransferases / Q000032:analysis; D006031:Glycosylation; D008364:Mannosyltransferases / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis; D008861:Microsomes / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D009844:Oligosaccharides / Q000096:biosynthesis; D046988:Proteasome Endopeptidase Complex / Q000032:analysis; D019704:Protein Disulfide-Isomerases / Q000032:analysis; D015800:Protozoan Proteins / Q000378:metabolism*; D000069816:SEC Translocation Channels
D004286:Dolichols; D008565:Membrane Proteins; D009844:Oligosaccharides; D015800:Protozoan Proteins; D000069816:SEC Translocation Channels; D005964:Glucosyltransferases; D008364:Mannosyltransferases; C054676:dolichol phosphate glucose-dolichol diphosphate oligosaccharide glucosyltransferase; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D000135:Acid Phosphatase; D000118:Acetylglucosaminidase; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases