Flotillin-1 interacts with the serotonin transporter and modulates chronic corticosterone response.

Aberrant serotonergic neurotransmission in the brain is considered at the core of the pathophysiological mechanisms involved in neuropsychiatric disorders. Gene by environment interactions contribute to the development of depression and involve modulation of the availability and functional activity of the serotonin transporter (SERT). Using behavioral and in vivo electrophysiological approaches together with biochemical, molecular-biological and molecular imaging tools we establish Flotillin-1 (Flot1) as a novel protein interacting with SERT and demonstrate its involvement in the response to chronic corticosterone (CORT) treatment. We show that genetic Flot1 depletion augments chronic CORT-induced behavioral despair and describe concomitant alterations in the expression of SERT, activity of serotonergic neurons and alterations of the glucocorticoid receptor transport machinery. Hence, we propose a role for Flot1 as modulatory factor for the depressogenic consequences of chronic CORT exposure and suggest Flotillin-1-dependent regulation of SERT expression and activity of serotonergic neurotransmission at the core of the molecular mechanisms involved.

BioID identifies proteins involved in the cell biology of caveolae.

The mechanisms controlling the abundance and sub-cellular distribution of caveolae are not well described. A first step towards determining such mechanisms would be identification of relevant proteins that interact with known components of caveolae. Here, we applied proximity biotinylation (BioID) to identify a list of proteins that may interact with the caveolar protein cavin1. Screening of these candidates using siRNA to reduce their expression revealed that one of them, CSDE1, regulates the levels of mRNAs and protein expression for multiple components of caveolae. A second candidate, CD2AP, co-precipitated with cavin1. Caveolar proteins were observed in characteristic and previously un-described linear arrays adjacent to cell-cell junctions in both MDCK cells, and in HeLa cells overexpressing an active form of the small GTPase Rac1. CD2AP was required for the recruitment of caveolar proteins to these linear arrays. We conclude that BioID will be useful in identification of new proteins involved in the cell biology of caveolae, and that interaction between CD2AP and cavin1 may have an important role in regulating the sub-cellular distribution of caveolae.

Cells respond to deletion of CAV1 by increasing synthesis of extracellular matrix.

A range of cellular functions have been attributed to caveolae, flask-like invaginations of the plasma membrane. Here, we have used RNA-seq to achieve quantitative transcriptional profiling of primary embryonic fibroblasts from caveolin 1 knockout mice (CAV1-/- MEFs), and thereby to gain hypothesis-free insight into how these cells respond to the absence of caveolae. Components of the extracellular matrix were decisively over-represented within the set of genes displaying altered expression in CAV1-/- MEFs when compared to congenic wild-type controls. This was confirmed biochemically and by imaging for selected examples. Up-regulation of components of the extracellular matrix was also observed in a second cell line, NIH-3T3 cells genome edited to delete CAV1. Up-regulation of components of the extracellular matrix was detected in vivo by assessing collagen deposition and compliance of CAV1-/- lungs. We discuss the implications of these findings in terms of the cellular function of caveolae.

Distribution of lipid raft markers in live cells.

GPI (glycosylphosphatidylinositol)-anchored proteins are characteristic components of biochemically defined lipid rafts. Rafts may be involved in T-cell stimulation, but it is not clear whether molecules involved in TCR (T-cell receptor) signalling are partitioned to T-cell synapses through raft microdomains or through specific protein-protein interactions. We have used FRET (fluorescence resonance energy transfer) analysis to study the distribution of GPI-anchored fluorescent proteins in the plasma membrane of live cells. Multiple criteria suggested that FRET between different GPI-anchored fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, unclustered distribution. Stimulation of TCR signalling in Jurkat T-cells by beads coated with antibodies against TCR subunits resulted in localized increases in fluorescence of raft markers. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of raft markers in these regions.

GM1-containing lipid rafts are depleted within clathrin-coated pits.

Recent studies show that markers for lipid rafts are among the plasma membrane components most likely to be internalized independently of clathrin-coated pits, and there is evidence to suggest that lipid rafts may play a functional role in endocytic trafficking [1-5]. However, lipid rafts themselves are commonly defined purely in biochemical terms, by resistance to detergent extraction. The existence of rafts in live-cell membranes remains controversial [6-8], and their distribution relative to endocytic machinery has not been investigated. This study employs fluorescence resonance energy transfer (FRET) to show that in the plasma membrane (PM) of living cells the glycosphingolipid GM1, labeled with cholera toxin B subunit (CTB) [9,10], is found at least in part within clusters that also include GPI-linked proteins. These clusters are cholesterol-dependent and exclude non-raft proteins such as transferrin receptor and so possess predicted properties of lipid rafts. This type of lipid raft is largely excluded from clathrin-positive regions of the PM. They are found within Caveolin-positive regions at the same concentration as at the rest of the cell surface. The data provide evidence for a model in which lipid rafts are distributed uniformly across most of the PM of nonpolarized cells but are prevented from entering clathrin-coated pits.

Endocytosis without clathrin coats.

Endocytosis is involved in an enormous variety of cellular processes. To date, most studies on endocytosis in mammalian cells have focused on pathways that start with uptake through clathrin-coated pits. Recently, new techniques and reagents have allowed a wider range of endocytic pathways to begin to be characterized. Various non-clathrin endocytic mechanisms have been identified, including uptake through caveolae, macropinosomes and via a separate constitutive pathway. Many markers for clathrin-independent endocytosis are found in detergent-resistant membrane fractions, or lipid rafts. We will discuss these emerging new findings and their implications for the nature of lipid rafts themselves, as well as for the potential roles of non-clathrin endocytic pathways in remodeling of the plasma membrane and in regulating the membrane composition of specific intracellular organelles.

Rapid cycling of lipid raft markers between the cell surface and Golgi complex.

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.

Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome-Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins.

Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.

The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast.

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.

SNAREs and membrane fusion in the Golgi apparatus.

Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell. They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs. Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion. NSF is an ATPase which will disrupt complexes composed of different SNAREs. However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane. The essential role of NSF may be to prime SNAREs for a direct role during fusion. The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi. The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar. Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes. Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles. In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue. There are only eight members of the syntaxin family in yeast. Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex. They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi. We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles.

A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion.

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

Two syntaxin homologues in the TGN/endosomal system of yeast.

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v-SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra-Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.

The Sec1p homologue Vps45p binds to the syntaxin Tlg2p.

SNAREs are compartmentally specific membrane proteins required for intracellular membrane fusion. Homologues of the Saccharomyces cerevisiae protein Sec1p interact with, and are likely to be involved in regulation of, the syntaxin family of SNAREs. In yeast there are 7 functionally distinct syntaxins but only four clearly identifiable homologues of Sec1p. One of these, Vps45p, is required for transport from Golgi to late endosomes, and has been implicated in the function of the late endosomal syntaxin Pep12p. However, there is evidence that not all the functions of Pep12p are equally dependent on Vps45p, and conversely that the phenotypes of vps45 mutants cannot be explained entirely by loss of Pep12p activity. We have recently characterised two yeast syntaxins which function in trans-Golgi or endosomal compartments, Tlg1p and Tlg2p. We show here that the principal binding site for Vps45p on intracellular membranes is provided by Tlg2p rather than Pep12p, and that Vps45p is required for stable expression of Tlg2p. Vps45p is also associated with Tlg1p as part of a triple complex containing both Tlg1p and Tlg2p. Since a deltavps45 deltatlg2 double mutant has a more severe vacuolar protein sorting defect than a deltatlg2 mutant, Vps45p cannot only interact with Tlg2p. It appears that the role of Vps45p in protein traffic is more complex than has previously been assumed.

Homotypic vacuolar fusion mediated by t- and v-SNAREs.

Membrane fusion is necessary both in the eukaryotic secretory pathway and for the inheritance of organelles during the cell cycle. In the secretory pathway, heterotypic fusion takes place between small transport vesicles and organelles. It requires N-ethylmaleimide-sensitive fusion protein (NSF/Sec18p), soluble NSF attachment proteins (SNAPs/Sec17p) and SNAP receptors (SNAREs). SNAREs are integral membrane proteins (v-SNAREs on vesicles, t-SNAREs on the target organelles) and are thought to provide specificity to the fusion process. It has been suggested that Sec17p and Sec18p bind to v-SNARE/t-SNARE complexes and mediate the membrane fusion event. Homotypic fusion of yeast vacuoles also requires Sec17p and Sec18p (ref. 6), but in vitro they are needed only to 'prime' the vacuoles, not for subsequent docking or fusion. It has been unclear whether these reactions involve SNAREs that are similar to those previously identified in heterotypic fusion systems and, hence, whether the actions of Sec18p/NSF and Sec17p/alpha SNAP in these systems can be compared. Here we identify typical v- and t-SNAREs on the yeast vacuolar membrane. Although both are normally present, vacuoles containing only the v-SNARE can fuse with those containing only the t-SNARE. Vacuoles containing neither SNARE cannot fuse with those containing both, demonstrating that docking is mediated by cognate SNAREs on the two organelle membranes. Even when t- and v-SNAREs are on separate membranes, Sec17p and Sec18p act at the priming stage. Their action is not required at the point of assembly of the SNARE complex, nor for the fusion event itself.

Molecular cloning and deduced amino acid sequences of the alpha- and beta- subunits of mammalian NAD(+)-isocitrate dehydrogenase.

A 153 bp fragment of the cDNA encoding the beta-subunit of pig heart NAD(+)-isocitrate dehydrogenase (NAD(+)-ICDH) was specifically amplified by PCR, using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate cDNA clones encoding the complete mature form of the beta-subunit from a monkey testis cDNA library. Examination of the deduced amino acid sequence of the monkey subunit and the partial sequence of the pig heart enzyme revealed a high level of sequence conservation. In addition, 3 overlapping fragments of the cDNA for the alpha-subunit of monkey NAD(+)-ICDH were amplified using oligonucleotide primers derived from the cDNA sequence of a subunit of bovine NAD(+)-ICDH (EMBL accession no: U07980). These cDNA fragments allow deduction of the amino acid sequence of the alpha-subunit. Since the gamma-subunit of monkey NAD(+)-ICDH has already been cloned [Nichols, Hall, Perry and Denton (1993) Biochem. J. 295, 347-350], a deduced amino acid sequence is now available for all three subunits of mammalian NAD(+)-ICDH. Interrelationships between these subunits are discussed and they are compared with the two subunits of yeast NAD(+)-ICDH and Escherichia coli NADP(+)-ICDH.

Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions.

In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other three dehydrogenases may involve separate calcium binding subunits.

Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart.

The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available.

Molecular cloning and deduced amino acid sequences of the gamma-subunits of rat and monkey NAD(+)-isocitrate dehydrogenases.

A 600 bp cDNA fragment encoding part of the gamma-subunit of pig heart NAD(+)-isocitrate dehydrogenase (ICDH gamma) was amplified by PCR using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate clones encoding the complete mature forms of the gamma-subunit from rat epididymis and monkey testis cDNA libraries. Comparison of the deduced amino acid sequences of the rat and monkey subunits and the partial sequence of the pig heart enzyme revealed a remarkably high level of sequence identity. The relationship between the deduced amino acid sequences of the NAD(+)-ICDH gamma-subunits and those of nonmammalian NAD(+)- and NADP(+)-ICDH subunits is discussed.