Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins

A rotating annulus driven by localized convective forcing: a new atmosphere-like experiment

We present an experimental study of flows in a cylindrical rotating annulus convectively forced by local heating in an annular ring at the bottom near the external wall and via a cooled circular disk near the axis at the top surface of the annulus. This new configuration is distinct from the classical thermally-driven annulus analogue of the atmosphere circulation, in which thermal forcing is applied uniformly on the sidewalls, but with a similar aim to investigate the baroclinic instability of a rotating, stratified flow subject to zonally symmetric forcing. Two vertically and horizontally displaced heat sources/sinks are arranged so that, in the absence of background rotation, statically unstable Rayleigh-Bénard convection would be induced above the source and beneath the sink, thereby relaxing strong constraints placed on background temperature gradients in previous experimental configurations based on the conventional rotating annulus. This better emulates local vigorous convection in the tropics and polar regions of the atmosphere whilst also allowing stably-stratified baroclinic motion in the central zone of the annulus, as in midlatitude regions in the Earth’s atmosphere. Regimes of flow are identified, depending mainly upon control parameters that in turn depend on rotation rate and the strength of differential heating. Several regimes exhibit baroclinically unstable flows which are qualitatively similar to those previously observed in the classical thermally-driven annulus, However, in contrast to the classical configuration, they typically exhibit more spatiotemporal complexity. Thus, several regimes of flow demonstrate the equilibrated co-existence of, and interaction between, free convection and baroclinic wave modes. These new features were not previously observed in the classical annulus and validate the new setup as a tool for exploring fundamental atmosphere-like dynamics in a more realistic framework. Thermal structure in the fluid is investigated and found to be qualitatively consistent with previous numerical results, with nearly isothermal conditions respectively above and below the heat source and sink, and stably-stratified, sloping isotherms in the near-adiabatic interior