Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Protein-mediated surface structuring in biomembranes.

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism.

c-Fos, a transcription factor that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription factor activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

c-Fos is surface active and interacts differentially with phospholipid monolayers.

The transcription factor c-Fos forms stable Gibbs and Langmuir monolayers at the air-buffer interface. Its marked surface activity is enhanced by penetration into phospholipid films above the protein's own maximum adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid polar head group and the increases of lateral surface pressure generated are comparable to those of membrane-active proteins. The surface activity of c-Fos is strong enough to thermodynamically drive and retain c-Fos at the membrane interface where it may exert direct or indirect effects.

Mapping chromosome landmarks in the centromere I region of Neurospora crassa.

Chromosome translocation breakpoints, RFLP heterozygosity in partial chromosome duplications, and RFLP-marked crossover events have been used as chromosomal landmarks to find the position and orientation of cloned regions flanking centromere I of Neurospora crassa. Determination of physical:genetic ratios in genomic regions flanking the loci mei-3, un-2, and his-2 supports previous evidence indicating that recombinational activity is lower in regions flanking centromere I than in the general N. crassa genome. The homogeneous distribution of crossover events found in these regions suggests that there is not a gradient of crossover inhibition in the vicinity of centromere I. Thus, a largely extended centromeric effect and/or a general crossover inhibitory effect operating on linkage group I (LGI) could constitute the basis of these abnormal physical:genetic ratios. A DNA element containing about 76% A+T was isolated from the centromeric end of a cloned region on LGIR. The fragment includes a previously undescribed DNA sequence, highly repeated in the Neurospora genome, which may correspond to centromeric DNA.