Helmut Krämer

The three operators of the lac operon cooperate in repression

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one ‘pseudo‐operator’ O2 or O3 decreases repression by wild‐type tetrameric Lac repressor approximately 2‐ to 3‐fold; absence of both ‘pseudo‐operators’ decreases repression greater than 50‐fold. O1 alone represses under these conditions only approximately 20‐fold. Dimeric active Lac repressor (iadi) represses the wild‐type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non‐denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a ‘tandem’ structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a ‘sandwich’ structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.

A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

The bride of sevenless and sevenless interaction: Internalization of a transmembrane ligand

During Drosophila retinal development, the R8 photo-receptor neuron induces a neighboring cell to assume an R7 cell fate through cell contact. This is mediated by the transmembrane protein bride of sevenless (boss) on the surface of the R8 cell, which binds the sevenless tyrosine kinase receptor (sev) on the surface of the R7 precursor cell. The boss protein, which contains a large extracellular domain, seven transmembrane segments, and a C-terminal cytoplasmic domain, has an exceptional structure for a ligand of a receptor tyrosine kinase. Using a panel of antibodies directed to various cytoplasmic and extracellular epitopes, we demonstrate that the entire boss protein from its extreme N-terminus to its extreme C-terminus is internalized by sev-expressing tissue culture cells and by the R7 precursor cell in the developing eye imaginal disc. The receptor-mediated transfer of a transmembrane ligand represents a novel mechanism for protein transfer between developing cells.

Induction in the developing compound eye of Drosophila: Multiple mechanisms restrict R7 induction to a single retinal precursor cell

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.

Not just pretty eyes: Drosophila eye-colour mutations and lysosomal delivery

Analysis of Drosophila eye-colour mutations has made seminal contributions to the fields of genetics and biochemistry. Recent findings suggest that a subset of eye-colour genes is crucial for vesicular transport of proteins to pigment granules, specialized lysosomes of eye-pigment cells. Thus, classical work describing more than 85 eye-colour mutations and their genetic interactions offers a remarkable, untapped resource for the genetic analysis of protein delivery to lysosomes.

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Interaction of the autophagosomal SNARE Syntaxin 17 (Syx17) with the homotypic fusion and vacuole protein–sorting (HOPS) tethering complex is necessary for the fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic degradation and biosynthetic transport to lysosomes and eye pigment granules.

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/‐ 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = −0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle‐repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non‐integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.

Microtubule-dependent endosomal sorting of clathrin-independent cargo by Hook1

Hook1, a microtubule and cargo tethering protein, is important for the sorting of clathrin-independent cargoes away from EEA1+ endosomes and promotes their recycling.