Muriel Mari

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.A reservoir of Atg9-containing vesicles and tubules provides the initial membranes necessary for autophagophore formation in yeast.

Regulation of endoplasmic reticulum turnover by selective autophagy

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy (‘ER-phagy’). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae

Cells store metabolic energy in the form of neutral lipids that are deposited within lipid droplets (LDs). In this study, we examine the biogenesis of LDs and the transport of integral membrane proteins from the endoplasmic reticulum (ER) to newly formed LDs. In cells that lack LDs, otherwise LD-localized membrane proteins are homogenously distributed in the ER membrane. Under these conditions, transcriptional induction of a diacylglycerol acyltransferase that catalyzes the formation of the storage lipid triacylglycerol (TAG), Lro1, is sufficient to drive LD formation. Newly formed LDs originate from the ER membrane where they become decorated by marker proteins. Induction of LDs by expression of the second TAG-synthesizing integral membrane protein, Dga1, reveals that Dga1 itself moves from the ER membrane to concentrate on LDs. Photobleaching experiments (FRAP) indicate that relocation of membrane proteins from the ER to LDs is independent of temperature and energy, and thus not mediated by classical vesicular transport routes. LD-localized membrane proteins are homogenously distributed at the perimeter of LDs, they are free to move over the LD surface and can even relocate back into the ER, indicating that they are not restricted to specialized sites on LDs. These observations indicate that LDs are functionally connected to the ER membrane and that this connection allows the efficient partitioning of membrane proteins between the two compartments.

Sorting by the Cytoplasmic Domain of the Amyloid Precursor Protein Binding Receptor SorLA

ABSTRACT SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimers disease-associated Aβ-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLAs trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 μ1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLAs transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors

Mannose‐6‐phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi‐ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno‐electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin‐coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)‐positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1‐dependent carriers, which we named endosome‐to‐TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three‐dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE‐derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.

CD2AP/CMS Regulates Endosome Morphology and Traffic to the Degradative Pathway Through its Interaction with Rab4 and c‐Cbl

The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4‐Q67L, the GTP‐bound form of Rab4. We cloned an ubiquitous 80‐kDa protein, identical to CD2‐associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4‐Q67L in the yeast two‐hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS‐positive vesicles. However, coexpression of Rab4‐Q67L with CD2AP/CMS induces a significant enlargement of EEA1‐positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c‐Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c‐Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c‐Cbl inhibits ligand‐induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c‐Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.

A role for Atg8–PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8–PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8–PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8–PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

The puzzling origin of the autophagosomal membrane

Autophagy is one of the newest and fastest emerging research areas in biomedical life sciences. Autophagosomes, large double-membrane vesicles enclosing cytoplasmic components targeted for degradation, are the hallmark of this catabolic pathway. The origin of the lipid bilayers composing these transport carriers has been the central enigma of the field since the discovery of autophagy. A series of recent studies has implicated several cellular organelles as the possible source of the autophagosomal membranes, if anything further clouding our view. In this compendium, we will discuss these apparently contradictory results and briefly emphasize the relevance of determining the lipid source used for autophagy for future translational research, for example in drug discovery programs.

Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif.