Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis.
Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.
Tomaska
L
L
Lineberger Comprehensive Cancer Center and Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599, USA.
Makhov
A M
AM
Nosek
J
J
Kucejova
B
B
Griffith
J D
JD
D017104:Bacteriophage M13 / Q000235:genetics; D015153:Blotting, Western; D002175:Candida / Q000737:chemistry* / Q000166:cytology / Q000235:genetics; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D008793:Metrizamide; D008854:Microscopy, Electron; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016615:Telomere / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008793:Metrizamide