MAP-1 and IAP-1, Two Novel AAA Proteases with Catalytic Sites on Opposite Membrane Surfaces in Mitochondrial Inner Membrane ofNeurospora crassa

Abstract

Eukaryotic AAA proteases form a conserved family of membrane-embedded ATP-dependent proteases but have been analyzed functionally only in the yeast Saccharomyces cerevisiae. Here, we have identified two novel members of this protein family in the filamentous fungus Neurospora crassa, which were termed MAP-1 and IAP-1. Both proteins are localized to the inner membrane of mitochondria. They are part of two similar-sized high molecular mass complexes, but expose their catalytic sites to opposite membrane surfaces, namely, the intermembrane and the matrix space. Disruption of iap-1 by repeat-induced point mutation caused a slow growth phenotype at high temperature and stabilization of a misfolded inner membrane protein against degradation. IAP-1 could partially substitute for functions of its yeast homolog Yme1, demonstrating functional conservation. However, respiratory growth at 37 degrees C was not restored. Our results identify two components of the quality control system of the mitochondrial inner membrane in N. crassa and suggest that AAA proteases with catalytic sites exposed to opposite membrane surfaces are present in mitochondria of all eukaryotic cells.

Unmasking a temperature-dependent effect of theP. anserinai-AAA protease on aging and development

“…Moreover, IAP-1 can only partially substitute YME1 in S. cerevisiae, pointing to subtle differences in the function of the two i-AAA proteases. 15,16 Very little is known about the human homolog YME1L1. Interestingly, YME1L1 is able to rescue the temperature-sensitive phenotype of Yme1-deficient yeast cells underlining the conserved functions of i-AAA proteases.…”

“…15,47 At 37°C, Yme1 deletion strains of yeast are unable to grow on non-fermentable carbon sources but do so at 14°C. On rich medium containing P. anserina is not comparable to chronological aging in yeast, which is a model of aging of post-mitotic cells.…”

“…In both fungi, deletion of the corresponding genes leads to impaired growth at high temperatures. 15 In contrast to yeast, decreased temperatures do the i-AAA protease in heat stress and identify the corresponding pathway as relevant for aging and development.…”

Substrate Recognition by AAA+ ATPases: Distinct Substrate Binding Modes in ATP-Dependent Protease Yme1 of the Mitochondrial Intermembrane Space

“…DNA fragments coding for AAA protease domains to be introduced were amplified by PCR using primer pairs harboring homologous sequences (40 bp) flanking the integration site. ⌬yme1 cells were cotransformed with these PCR-amplified DNA fragments and linearized with pVT100U-YME1 or pVT100U-IAP-1 (20).…”

“…All strains used in this study are derivatives of W303. YME1 and IAP-1 genes and derivatives thereof were expressed in a ⌬yme1 yeast strain described previously (YCK10 [20]). Yme1 and hybrid proteins accumulated at similar levels in the different strains as revealed by immunoblotting of cellular extracts using Yme1-specific antibodies (data not shown).…”