Search

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.

Interactions between yeast Dnm1p, Mdv1p, and Fis1p are required to form fission complexes that catalyze division of the mitochondrial compartment. During the formation of mitochondrial fission complexes, the Dnm1p GTPase self-assembles into large multimeric complexes on the outer mitochondrial membrane that are visualized as punctate structures by fluorescent labeling. Although it is clear that Fis1p.Mdv1p complexes on mitochondria are required for the initial recruitment of Dnm1p, it is not clear whether Dnm1p puncta assemble before or after this recruitment step. Here we show that the minimum oligomeric form of cytoplasmic Dnm1p is a dimer. The middle domain mutant protein Dnm1G385Dp forms dimers in vivo but fails to assemble into punctate structures. However, this dimeric mutant stably interacts with Mdv1p on the outer mitochondrial membrane, demonstrating that assembly of stable Dnm1p multimers is not required for Dnm1p-Mdv1p association or for mitochondrial recruitment of Dnm1p. Dnm1G385Dp is reported to be a terminal dimer in vitro. We describe conditions that allow assembly of Dnm1G385Dp into functional fission complexes on mitochondria in vivo. Using these conditions, we demonstrate that multimerization of Dnm1p is required to promote reorganization of Mdv1p from a uniform mitochondrial localization into punctate fission complexes. Our studies also reveal that Fis1p is present in these assembled fission complexes. Based on our results, we propose that Dnm1p dimers are initially recruited to the membrane via interaction with Mdv1p.Fis1p complexes. These dimers then assemble into multimers that subsequently promote the reorganization of Mdv1p into punctate fission complexes.

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.

In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA). Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the matrix, raising the possibility that this protein plays a direct role in organizing, replicating, or segregating mtDNA. Although Mmm1p has been shown to cross the outer membrane with its C terminus facing the cytoplasm, the location of the N terminus has not been resolved. Here we show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N terminus to the matrix. Surprisingly, deletion of the N-terminal extension decreased steady-state levels of the Mmm1 protein but did not affect mitochondrial morphology or mtDNA maintenance. Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long N-terminal extension, substituted for loss of Mmm1p in budding yeast. These results indicate that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance. Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p are required for foci formation and mitochondrial targeting, respectively. Our data suggest that the double membrane-spanning topology of Mmm1p at the membrane contact site is critical for formation of tubular mitochondria.

In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

The biosynthesis of glucuronosyl diacylglycerol from UDP-glucuronate and diacylglycerol is catalyzed by an enzyme found in both the 34,800 X g supernatant and particulate preparations from disrupted Pseudomonas diminuta (ATCC 11586). UDP-glucuronate served as the glucuronosyl donor and could not be replaced by glucuronic acid, glucuronate-1-phosphate, and a number of nucleotide-linked sugars. The maximum velocity was estimated to be 19 nmol of glucuronosyl diacylglycerol synthesized/h/mg of protein in the presence of the 34,800 X g particulate enzyme and 63 nmol/h/mg of protein with the 34,800 X g supernatant preparation. The apparent Km for UDP-glucuronate was 4.2 micronM for supernatant and 4.4 to 6.0 micronM for particulate preparations. The biosynthesis of glucuronosyl diacylglycerol in vitro, was strongly dependent upon exogenous diacylglycerols containing unsaturated and shorter chain fatty acids. The enzymatic activity was very heat-labile and lost about 80% of the initial rate of synthesis after preincubation for 5 min at 37 degrees. The reaction was stimulated by 14.7 mM Triton X-100 and had an optimal pH of 7.1 and an ionic strength of 0.2 M. Divalent cations were not required.