J. Paul Luzio

Lysosomes: fusion and function

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

Lysine‐63‐linked ubiquitination is required for endolysosomal degradation of class I molecules

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposis sarcoma‐associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3‐associated class I by lysine‐63‐linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine‐63‐linked polyubiquitination requires both UbcH5b/c and Ubc13‐conjugating enzymes for initiating mono‐ and subsequent polyubiquitination of class I, and the clathrin‐dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine‐63‐linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.

Myosin VI isoform localized to clathrin‐coated vesicles with a role in clathrin‐mediated endocytosis

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co‐localizing with clathrin‐ coated pits/vesicles. Using full‐length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) we have shown that myosin VI associates and co‐localizes with clathrin‐coated pits/vesicles by its C‐terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)‐2 and clathrin, and enriched in purified clathrin‐coated vesicles. Over‐expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin‐coated pits/vesicles and shown to modulate clathrin‐mediated endocytosis.

Myosin VI Binds to and Localises with Dab2, Potentially Linking Receptor‐Mediated Endocytosis and the Actin Cytoskeleton

Myosin VI, an actin‐based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two‐hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin‐coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low‐density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP‐2, whereas a C‐terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin‐coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.

Late endosomes: sorting and partitioning in multivesicular bodies.

Late endosomes, which have the morphological characteristics of multivesicular bodies, have received relatively little attention in comparison with early endosomes and lysosomes. Recent work in mammalian and yeast cells has given insights into their structure and function, including the generation of their multivesicular morphology. Lipid partitioning to create microdomains enriched in specific lipids is observed in late endosomes, with some lumenal vesicles enriched in lysobisphosphatidic acid and others in phosphatidylinositol 3‐phosphate. Sorting of membrane proteins into the lumenal vesicles may occur because of the properties of their trans‐membrane domains, or as a result of tagging with ubiquitin. Yeast class E Vps proteins and their mammalian orthologs are the best candidates to make up the protein machinery that controls inward budding, a process that starts in early endosomes. Late endosomes are able to undergo homotypic fusion events and also heterotypic fusion with lysosomes, a process that delivers endocytosed macromolecules for proteolytic degradation.

Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers.

Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pyloricauses the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker. Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments. This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles. The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans‐SNARE complex consisting of Q‐SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R‐SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell‐free systems, we confirmed this result, but found that the same Q‐SNAREs can combine with an alternative R‐SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co‐immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N‐terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

Protein–Protein Interactions of ESCRT Complexes in the Yeast Saccharomyces cerevisiae

Ten class E Vps proteins in yeast are known components of the ESCRT complexes I, II and III, which are required for the sorting of proteins to the lumenal membranes of multivesicular bodies. We used the yeast 2 hybrid system to analyze the protein–protein interactions of all 17 soluble class E Vps proteins, as well as proteins thought to be required for the ubiquitination and deubiquitination of cargo proteins at multivesicular bodies. We identified novel interactions between yeast ESCRT complex components suggesting that ESCRTI binds to both ESCRTII and ESCRTIII. These interactions were confirmed by GST pull‐down experiments. Our data indicate that the link between ESCRTI and ESCRTIII is via Vps28p and Vps37p/Srn2p binding directly to Vps20p, as well as through indirect interactions via ESCRTII. This is in contrast to the situation in mammalian cells where ESCRTI and ESCRTIII interact indirectly via ALIX, the mammalian homologue of yeast proteins Vps31p/Bro1p and Rim20p. Our data also enable us to link all soluble class E Vps proteins to the ESCRT complexes. We propose the formation of a large multimeric complex on the endosome membrane consisting of ESCRTI, ESCRTII, ESCRTIII and other associated proteins.

Degradation of Endocytosed Epidermal Growth Factor and Virally Ubiquitinated Major Histocompatibility Complex Class I Is Independent of Mammalian ESCRTII

Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These data suggest the essential role of four ESCRT complexes in multivesicular body protein sorting. However, the putative mammalian ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional role in endosomal transport. We have characterized the human ESCRTII complex and investigated its function in endosomal trafficking. The human ESCRTII proteins interact with one another, with hVps20p (a component of ESCRTIII), and with their yeast homologues. Our interaction data from yeast two-hybrid studies along with experiments with purified proteins suggest an essential role for the N-terminal domain of hVps22p in the formation of a heterotetrameric ESCRTII complex. Although human ESCRTII is found in the cytoplasm and in the nucleus, it can be recruited to endosomes upon overexpression of dominant-negative hVps4Bp. Interestingly, we find that small interference RNA depletion of mammalian ESCRTII does not affect degradation of epidermal growth factor, a known cargo of the multivesicular body protein sorting pathway. We also show that depletion of the deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)) and UBPY (ubiquitin isopeptidase Y) have opposite effects on epidermal growth factor degradation, with UBPY depletion causing dramatic swelling of endosomes. Down-regulation of another cargo, the major histocompatibility complex class I in cells expressing the Kaposi sarcoma-associated herpesvirus protein K3, is unaffected in ESCRTII-depleted cells. Our data suggest that mammalian ESCRTII may be redundant, cargo-specific, or not required for protein sorting at the multivesicular body.