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The trans-Golgi network coordinates sorting and secretion of proteins and lipids to cellular organelles and the plasma membrane. It is revealed that Sdc1, a heparin sulfated proteoglycan present on basolateral membranes of polarized epithelia, requires the golgin GOPC for basolateral targeting, in contrast to its sorting requirements in nonpolarized cells.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome-to-Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.

Synaptobrevin/vesicle-associated membrane protein 2 (VAMP2) is an essential soluble N-ethyl maleimide–sensitive factor attachment protein receptor (SNARE) protein that has been extensively studied in its role in synaptic vesicle fusion. However, sorting and trafficking of VAMP2 within the endosomal system is not well understood. Here, we use the yeast VAMP2 homologue Snc1 to investigate the pathways and signals required for endocytic trafficking. We identify two genetically distinct retrieval pathways from the endosomal system: a plasma membrane recycling pathway that requires the Rcy1 F-box protein and a retrograde pathway originating from the multivesicular/prevacuole endosome dependent on the Snx4-Atg20 sorting nexin complex. Lysine residues within the transmembrane domain of Snc1 are necessary for presentation of a Snx4-Atg20–dependent sorting signal located within its juxtamembrane region. Mutations of the transmembrane lysine residues ablate retrograde sorting and subject Snc1 to quality control via sorting into the degradative multivesicular endosome pathway. Degradative sorting requires lysine residues in the juxtamembrane region of Snc1 and is mediated by the Rsp5 ubiquitin ligase and its transmembrane adapters, Ear1 and Ssh4, which localize to endosome and vacuole membranes. This study shows that Snc1 is trafficked between the endosomal system and the Golgi apparatus via multiple pathways and provides evidence for protein quality control surveillance of a SNARE protein in the endo-vacuolar system.

Christopher G. Burd discusses recent studies that implicate the GRASP65 and GRASP55 proteins in organizing stacks of Golgi cisternae into a ribbon., Cisternae of the Golgi apparatus adhere to each other to form stacks, which are aligned side by side to form the Golgi ribbon. Two proteins, GRASP65 and GRASP55, previously implicated in stacking of cisternae, are shown to be required for the formation of the Golgi ribbon.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

Phosphatidylethanolamine is an abundant component of most cellular membranes whose physical and chemical properties modulate multiple aspects of organelle membrane dynamics. An evolutionarily ancient mechanism for producing phosphatidylethanolamine is to decarboxylate phosphatidylserine and the enzyme catalyzing this reaction, phosphatidylserine decarboxylase, localizes to the inner membrane of the mitochondrion. We characterize a second form of phosphatidylserine decarboxylase, termed PISD-LD, that is generated by alternative splicing of PISD pre-mRNA and localizes to lipid droplets and to mitochondria. Sub-cellular targeting is controlled by a common segment of PISD-LD that is distinct from the catalytic domain and is regulated by nutritional state. Growth conditions that promote neutral lipid storage in lipid droplets favors targeting to lipid droplets, while targeting to mitochondria is favored by conditions that promote consumption of lipid droplets. Depletion of both forms of phosphatidylserine decarboxylase impairs triacylglycerol synthesis when cells are challenged with free fatty acid, indicating a crucial role phosphatidylserine decarboxylase in neutral lipid storage. The results reveal a previously unappreciated role for phosphatidylserine decarboxylase in lipid droplet biogenesis.

Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)–retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Sphingolipids are structural components of organelle membranes that also participate in signal transduction pathways. Complex sphingolipids are trafficked from their site of synthesis in organelles of the early secretory pathway to the Golgi apparatus, the plasma membrane, and the endo-lysosomal system. We have developed fluorescence microscopy-based methods to monitor sphingolipid trafficking in coordination with secretory protein sorting. A sphingomyelin binding protein fused to a fluorescent protein, which we term "EQ-SM," is implemented to monitor sphingomyelin trafficking from the Golgi apparatus to the plasma membrane via secretory vesicles. A protocol is provided to determine if a query protein of interest is secreted from the cell via vesicles enriched in EQ-SM, an indication that the vesicle membrane is enriched in sphingomyelin. A complementary protocol is described that implements a chemically modified form of sphingosine, a metabolic precursor to complex sphingolipids, to visualize ceramide and complex sphingolipids in fixed cells. © 2018 by John Wiley & Sons, Inc.

Cargo-selective and nonselective autophagy pathways employ a common core autophagy machinery that directs biogenesis of an autophagosome that eventually fuses with the lysosome to mediate turnover of macromolecules. In yeast ( Saccharomyces cerevisiae) cells, several selective autophagy pathways fail in cells lacking the dimeric Snx4/Atg24 and Atg20/Snx42 sorting nexins containing a BAR domain (SNX-BARs), which function as coat proteins of endosome-derived retrograde transport carriers. It is unclear whether endosomal sorting by Snx4 proteins contributes to autophagy. Cells lacking Snx4 display a deficiency in starvation induced, nonselective autophagy that is severely exacerbated by ablation of mitochondrial phosphatidylethanolamine synthesis. Under these conditions, phosphatidylserine accumulates in the membranes of the endosome and vacuole, autophagy intermediates accumulate within the cytoplasm, and homotypic vacuole fusion is impaired. The Snx4-Atg20 dimer displays preference for binding and remodeling of phosphatidylserine-containing membrane in vitro, suggesting that Snx4-Atg20-coated carriers export phosphatidylserine-rich membrane from the endosome. Autophagy and vacuole fusion are restored by increasing phosphatidylethanolamine biosynthesis via alternative pathways, indicating that retrograde sorting by the Snx4 family sorting nexins maintains glycerophospholipid homeostasis required for autophagy and fusion competence of the vacuole membrane.

The yeast SNX4 sub-family of sorting nexin containing a Bin-Amphiphysin-Rvs domain (SNX-BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4-Atg20 and Snx4-Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat 3 distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.

Characterization of the structure of the N-terminal portion of yeast Sac1 in complex with the phosphatidylinositol 4-kinase effector Vps74 identifies how Sac1 is directed to dephosphorylate PtdIns4P in the Golgi., Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

Endosomal sorting and fission machineries act together to produce retrograde transport carriers., Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR–coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.

SummaryPlasma membrane PI4P helps determine the identity of this membrane and plays a key role in signal transduction as the precursor of PI(4,5)P2 and its metabolites. Here, we report the atomic structure of the protein scaffold that is required for the plasma membrane localization and function of Stt4/PI4KIIIα, the PI 4-kinase responsible for this PI4P pool. Both proteins of the scaffold, Efr3 and YPP1/TTC7, are composed of α-helical repeats, which are arranged into a rod in Efr3 and a superhelix in Ypp1. A conserved basic patch in Efr3, which binds acidic phospholipids, anchors the complex to the plasma membrane. Stt4/PI4KIIIα is recruited by interacting with the Ypp1 C-terminal lobe, which also binds to unstructured regions in the Efr3 C terminus. Phosphorylation of this Efr3 region counteracts Ypp1 binding, thus providing a mechanism through which Stt4/PI4KIIIα recruitment, and thus a metabolic reaction of fundamental importance in cell physiology, can be regulated.

We describe a regulatory mechanism that controls the activity of retromer, an evolutionarily conserved sorting device that orchestrates cargo export from the endosome. A spontaneously arising mutation that activates the yeast (Saccharomyces cerevisiae) CDC25 family phosphatase, Mih1, results in accelerated turnover of a subset of endocytosed plasma membrane proteins due to deficient sorting into a retromer-mediated recycling pathway. Mih1 directly modulates the phosphorylation state of the Vps26 retromer subunit; mutations engineered to mimic these states modulate the binding affinities of Vps26 for a retromer cargo, resulting in corresponding changes in cargo sorting at the endosome. The results suggest that a phosphorylation-based gating mechanism controls cargo selection by yeast retromer, and they establish a functional precedent for CDC25 protein phosphatases that lies outside of their canonical role in regulating cell cycle progression. DOI: http://dx.doi.org/10.7554/eLife.24126.001

Summary In the secretory pathway, budding of vesicular transport carriers from the trans-Golgi network (TGN) must coordinate specification of lipid composition with selection of secreted proteins. We elucidate a mechanism of soluble protein cargo sorting into secretory vesicles with a sphingomyelin-rich membrane; the integral membrane proteoglycan Syndecan-1 (SDC1) acts as a sorting receptor, capturing the soluble enzyme lipoprotein lipase (LPL) during export from the TGN. Sorting of LPL requires bivalent interactions between LPL and SDC1-linked heparan sulfate chains and between LPL and the Golgi membrane. Physical features of the SDC1 transmembrane domain, rather than a specific sequence, confer targeting of SDC1 and bound LPL into the sphingomyelin secretion pathway. This study establishes that physicochemical properties of a protein transmembrane domain that drive lateral heterogeneity of the plasma membrane also operate at the TGN to confer sorting of an integral membrane protein and its ligand within the biosynthetic secretory pathway.

The Drs2 flippase increases membrane curvature and anionic phospholipid composition of the membrane by flipping phosphatidylserine, which is critical for vesicular transport between the trans-Golgi network and early endosomes., Vesicle-mediated protein transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, protein transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for protein transport in this pathway are unknown. Here, we show that the Arf GTPase-activating protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular transport between the EE and the TGN.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.

Signaling by phosphatidylinositol 4-kinases (PI4Ks) in the Golgi apparatus controls lipid homeostasis and protein-sorting pathways. Signaling is shown to be terminated on the medial cisterna by a complex of a PI4K effector, Vps74, and Sac1, the major PtdIns4P phosphatase in the cell., In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.

Retromer complex mediates the sorting of cargo from the endosome to the Golgi apparatus. At the endosome, recognition of Ypt7 (Rab7) by the Vps35 retromer subunit is essential for the cargo export step of the retromer functional cycle. Retromer also controls Ypt7-regulated fusion dynamics of the late endovacuolar system., The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.

Bidirectional traffic between the Golgi apparatus and the endosomal system sustains the functions of the trans-Golgi network (TGN) in secretion and organelle biogenesis. Export of cargo from the TGN via anterograde trafficking pathways depletes the organelle of sorting receptors, processing proteases, SNARE molecules, and other factors, and these are subsequently retrieved from endosomes via the retrograde pathway. Recent studies indicate that retrograde trafficking is vital to early metazoan development, nutrient homeostasis, and for processes that protect against Alzheimer's and other neurological diseases.

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.

Summary How the principal functions of the Golgi apparatus—protein processing, lipid synthesis, and sorting of macromolecules—are integrated to constitute cargo-specific trafficking pathways originating from the trans-Golgi network (TGN) is unknown. Here, we show that the activity of the Golgi localized SPCA1 calcium pump couples sorting and export of secreted proteins to synthesis of new lipid in the TGN membrane. A secreted Ca2+-binding protein, Cab45, constitutes the core component of a Ca2+-dependent, oligomerization-driven sorting mechanism whereby secreted proteins bound to Cab45 are packaged into a TGN-derived vesicular carrier whose membrane is enriched in sphingomyelin, a lipid implicated in TGN-to-cell surface transport. SPCA1 activity is controlled by the sphingomyelin content of the TGN membrane, such that local sphingomyelin synthesis promotes Ca2+ flux into the lumen of the TGN, which drives secretory protein sorting and export, thereby establishing a protein- and lipid-specific secretion pathway.

Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.

Retention of Golgi-resident integral membrane proteins depends on the supply of PI(4)P., Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.

Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.

Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p–Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p–Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p–Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos.

Sorting of macromolecules within the endosomal system is vital for physiological control of nutrient homeostasis, cell motility, and proteostasis. Trafficking routes that export macromolecules from the endosome via vesicle and tubule transport carriers constitute plasma membrane recycling and retrograde endosome-to-Golgi pathways. Proteins of the sorting nexin family have been discovered to function at nearly every step of endosomal transport carrier biogenesis and it is becoming increasingly clear that they form the core machineries of cargo-specific transport pathways that are closely integrated with cellular physiology. Here, we summarize recent progress in elucidating the pathways that mediate the biogenesis of endosome-derived transport carriers.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm., Author Summary The human papillomaviruses are important carcinogens, but little is known about how these non-enveloped viruses traffic to the nucleus, the site of genome replication. We use imaging, biochemical, and genetic techniques to show that a multi-subunit intracellular trafficking machine known as retromer binds directly to the L2 minor capsid protein in the virus particle to initiate its transport from the endosome to other membrane-bound organelles farther inside the cell. Most notably, knock-down of retromer expression or mutation of newly identified retromer binding sites in L2 cause the accumulation of incoming HPV16 capsids in the endosome and prevent trafficking to the Golgi. These defects can be corrected by insertion of a retromer binding site from a cellular cargo. Because all previously known retromer cargoes are cellular transmembrane proteins, the virus represents a new class of retromer cargo. In addition to elucidating the mechanism of viral endosome escape, these results suggest that retromer may play a more versatile role in cell biology than previously recognized.

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns(3)P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns(3)P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns(3)P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns(3)P binding by the FYVE domain in vitro and in vivo . Another PtdIns(3)P-binding module, the PX domain of Vam7 and p40 phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns(3)P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.