Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation

The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.     

The ESCRT machinery: From the plasma membrane to endosomes and back again

Abstract

The manipulation and reorganization of lipid bilayers are required for diverse cellular processes, ranging from organelle biogenesis to cytokinetic abscission, and often involves transient membrane disruption. A set of membrane-associated proteins collectively known as the endosomal sorting complex required for transport (ESCRT) machinery has been implicated in membrane scission steps, which transform a single, continuous bilayer into two distinct bilayers, while simultaneously segregating cargo throughout the process. Components of the ESCRT pathway, which include 5 distinct protein complexes and an array of accessory factors, each serve discrete functions. This review focuses on the molecular mechanisms by which the ESCRT proteins facilitate cargo sequestration and membrane remodeling and highlights their unique roles in cellular homeostasis.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

Niemann-Pick type C mutations cause lipid traffic jam

The Niemann–Pick C protein (NPC1) is required for cholesterol transport from late endosomes and lysosomes to other cellular membranes. Mutations in NPC1 cause lysosomal lipid storage and progressive neurological degeneration. Cloning of the NPC1 gene has given us tools with which to investigate the function of this putative cholesterol transporter. Here, we discuss recent studies indicating that NPC1 is not a cholesterol-specific transport molecule. Instead, NPC1 appears to be required for the vesicular shuttling of both lipids and fluid-phase constituents from multivesicular late endosomes to destinations such as the trans -Golgi network.     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Intracellular distribution and late endosomal effects of the ocular albinism type 1 gene product: consequences of disease-causing mutations and implications for melanosome biogenesis

To investigate the function of ocular albinism type 1 ( OA1 ), the gene responsible for X-linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild-type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X-linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1-GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1-GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose-6-phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis.