The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.

Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.Cilia are microtubule-based organelles that function in motility, sensory reception, and signaling (1). Ciliary dysfunction results in numerous diseases and disorders commonly known as ciliopathies. Intraflagellar transport (IFT) is involved in cilium formation (2, 3) but also functions in other cellular processes, such as the recycling of Tcell receptors at the immune synapse (4). IFT relies on kinesin-2 and IFT-dynein molecular motors moving along the microtubule-based axoneme of cilia (5-7) and on the IFT complex, which contains at least 20 different protein subunits. Although ~600 proteins are known to reside in the cilium (8), we know very little about how they are recognized as ciliary cargo by the IFT machinery (9-11).To identify potential cargo-binding sites on the IFT complex, we carried out bioinformatical and biochemical screening and identified conserved domains that were not required for IFT complex formation. We reasoned that such domains could protrude from the IFT particlecore structure and would thus be in a prime position for cargo recognition. The two IFT core (12). Given that the cilium consists of a MT-based axoneme, IFT of large quantities of tubulin is required for cilium formation (13). We thus tested the tubulin-binding properties of HsIFT81N using affinity pull-downs ( Fig. 1D and fig. S4E) and microscale thermophoresis (MST) with unpolymerized bovine αβ-tubulin (Fig. 1, E S5F). Thus, the tubulin-binding module is formed by the IFT74/81 complex rather than by IFT81N alone.To dissect the binding mode in the IFT74/81:αβ-tubulin complex, samples were prepared from MT and unpolymerized αβ-tubulin lacking the highly acidic C-terminal tails, often referred to as E-hooks (12) ( fig. S5A). αβ-tubulin lacking E-hooks had similar affinity for IFT81N as intact tubulin ( fig. S5, B and C), which suggested that IFT81N recognizes the globular domain of αβ-tubulin with no substantial interaction with the E-hooks. IFT74/81 displayed robust MT binding in sedimentation assays, which was, however, reduced to background levels in the absence of the β-tubulin E-hook ( fig. S5E). Thus, IFT81N appears to bind the globular domain of tubulin to provide specificity, and IFT74N recognizes the β-tubulin tail to increase affinity (Fig. 1H).To examine the role of tubulin binding by IFT74/81 in a cellular system, we transiently expressed Flag-HsIFT81 or Flag-HsIFT81ΔN in human RPE-1 cells and induced formation ...

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.