The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC TPR ), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2 TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.kinesin | KLC | TPR domain | microtubule motor | cytoskeleton

The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt–Hoge–Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri‐nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi‐associated small GTPase Rab34. Rab34‐positive peri‐nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34‐induced peri‐nuclear lysosome clustering. FLCN interacts directly via its C‐terminal DENN domain with the Rab34 effector RILP. Using purified recombinant proteins, we show that the FLCN‐DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP. We propose a model whereby starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri‐nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell.

SummaryCytoplasmic dynein, the major motor driving retrograde axonal transport, must be actively localized to axon terminals. This localization is critical as dynein powers essential retrograde trafficking events required for neuronal survival, such as neurotrophic signaling. Here, we demonstrate that the outward transport of dynein from soma to axon terminal is driven by direct interactions with the anterograde motor kinesin-1. In developing neurons, we find that dynein dynamically cycles between neurites, following kinesin-1 and accumulating in the nascent axon coincident with axon specification. In established axons, dynein is constantly transported down the axon at slow axonal transport speeds; inhibition of the kinesin-1-dynein interaction effectively blocks this process. In vitro and live-imaging assays to investigate the underlying mechanism lead us to propose a new model for the slow axonal transport of cytosolic cargos, based on short-lived direct interactions of cargo with a highly processive anterograde motor.Video Abstract

Abstract. The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 #m 3, an inner membrane area of 6.5 #m 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of-7,500/#m 2 and occupy ~40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 ,~,) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.T HE molecular structure of mitochondria and in particular of their inner membrane is of great interest to bioenergetics because most of the enzymes involved in energy transduction are associated with these organelles (7). Many of the energy-transducing enzymes, notably the enzymes of the respiratory chain and the H÷-ATPase, are multisubunit complexes which span the membrane and represent ~80% of the total intrinsic protein mass of this membrane (21). Pictures of freeze-fractured inner membranes show a dense packing with intrinsic proteins (19,46). How dense the various protein complexes are built into the inner membrane, and how they are distributed along the plane of the membrane is important for the understanding of the mechanism of energy-transducing reactions in mitochondria.This sort of information is scarce due to the complexity of the mitochondrial ultrastructure and owing to the fact that it is not accessible through biochemical techniques alone. For instance, Gear and Bednarek (15) used a Coulter counter technique to estimate the number and volume of mitochondria in suspension and related these parameters to biochemical data. The Coulter counter method, however, measures only particle size, but cannot give information on the intramitochondrial structures relevant for bioenergetics, such as inner membrane and matrix. In this paper we present an approach which combines biochemical with stereological techniques so as to obtain parameters describing the molecular infrastructure of mitochondria and in particular of the inner membrane. Stereological procedures were applied on electron micrographs of purified preparations of rat liver mitochondria, to estimate mitochondrial number and volume, matrix volume, and surface areas of inner and outer memb...

No abstract