Biogenesis of the multifunctional lipid droplet: Lipids,proteins, and sites

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca²⁺ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca²⁺ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca²⁺ influx and secretory cargo sorting.     

ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1

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Highlights? ADF/cofilin localizes to the TGN and binds SPCA1 in an actin-dependent manner ? Dynamic actin regulates the activity of the Ca²⁺ pump SPCA1 ? ADF/cofilin facilitates secretory cargo sorting in a Ca²⁺-dependent manner     

Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca(2+) ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H(+) ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.     

Vesicular distribution of Secretory Pathway Ca<Superscript>2+</Superscript>-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn²⁺ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn²⁺ detoxification by excretion into bile. Although many pathways of cellular Mn²⁺ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn²⁺ detoxification by the Secretory Pathway Ca²⁺, Mn²⁺-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca²⁺-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn²⁺ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn²⁺. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn²⁺ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn²⁺ detoxification in liver.     

The Role of the Golgi-Resident SPCA Ca<Superscript>2+</Superscript>/Mn<Superscript>2+</Superscript> Pump in Ionic Homeostasis and Neural Function

Recent evidence highlights the functional importance of the Golgi apparatus (GA) in neurological diseases. The functions of the mammalian GA, in addition to the processing and transport of cargo, also include ionic homeostasis. Besides Ca²⁺-release channels which serves GA as an agonist-sensitive intracellular Ca²⁺ store, and Ca²⁺-binding proteins, the GA contains Ca²⁺-uptake mechanisms consisting of the well-known sarco-endoplasmic reticulum Ca²⁺-transport ATPases and the much less characterized secretory-pathway Ca²⁺-transport ATPases (SPCA). SPCA can transport both Ca²⁺ and Mn²⁺ into the Golgi lumen and therefore is involved in the cytosolic and intra-Golgi Ca²⁺ and Mn²⁺ homeostasis. It has shown that both of the mRNA and protein of SPCAs are highly expressed in brain. In addition, brain is the region with the highest activity of SPCA isoforms, which may be related to the involvement of Ca²⁺ and Mn²⁺ homeostasis in neural functions. In this review, we compile some recent findings showing that the SPCA isoform plays a much more important role in intracellular ionic homeostasis than previously anticipated and illustrating the involvement of SPCA isoforms in certain neurophysiological or neuropathological process. We are interested in gaining insight into the intricate role of the SPCA pumps to explain the GA-specific functions in neurological disorders.     

Receptor-mediated transport of vacuolar proteins: a critical analysis and a new model

In this article we challenge the widely accepted view that receptors for soluble vacuolar proteins (VSRs) bind to their ligands at the trans-Golgi network (TGN) and transport this cargo via clathrin-coated vesicles (CCV) to a multivesicular prevacuolar compartment. This notion, which we term the “classical model” for vacuolar protein sorting, further assumes that low pH in the prevacuolar compartment causes VSR–ligand dissociation, resulting in a retromer-mediated retrieval of the VSRs to the TGN. We have carefully evaluated the literature with respect to morphology and function of the compartments involved, localization of key components of the sorting machinery, and conclude that there is little direct evidence in its favour. Firstly, unlike mammalian cells where the sorting receptor for lysosomal hydrolases recognizes its ligand in the TGN, the available data suggests that in plants VSRs interact with vacuolar cargo ligands already in the endoplasmic reticulum. Secondly, the evidence supporting the packaging of VSR–ligand complexes into CCV at the TGN is not conclusive. Thirdly, the prevacuolar compartment appears to have a pH unsuitable for VSR–ligand dissociation and lacks the retromer core and the sorting nexins needed for VSR recycling. We present an alternative model for protein sorting in the TGN that draws attention to the much overlooked role of Ca²⁺ in VSR–ligand interactions and which may possibly also be a factor in the sequestration of secretory proteins.     

Synchronous intra-Golgi transport induces the release of Ca from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca²⁺) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca²⁺ concentrations in the cell cytosol ([Ca²⁺]_cyt) and inside the lumen of the Golgi apparatus ([Ca²⁺]_GA), we have revealed transient increases in [Ca²⁺]_cyt during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca²⁺]_GA restoration ability. Thus, this redistribution of Ca²⁺ from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca²⁺-dependent phase of SNARE-regulated fusion of Golgi compartments.     

SPCA2 Regulates Orai1 Trafficking and Store Independent Ca2+ Entry in a Model of Lactation

An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca²⁺-ATPase, and the Ca²⁺ influx channel Orai1, has previously been shown to contribute to elevated Ca²⁺ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca²⁺ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca²⁺ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca²⁺ entry (SICE), which mediates the massive basolateral Ca²⁺ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.     

The SPCA1 Ca2+ Pump and Intracellular Membrane Trafficking

The Golgi apparatus (GA) is a dynamic store of Ca²⁺ that can be released into the cell cytosol. It can thus participate in the regulation of the Ca²⁺ concentration in the cytosol ([Ca²⁺]_cyt), which might be critical for intra‐Golgi transport. Secretory pathway Ca²⁺‐ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca²⁺. The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca²⁺]_cyt that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis‐most and trans‐most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G‐protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra‐Golgi transport and maintenance of the Golgi ribbon.     

Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning

The trans-Golgi Network (TGN) sorts molecular “addresses” and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca²⁺ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.     

The Ca/H antiporter TMEM165 expression,localization in the developing,lactating and involuting mammary gland parallels the secretory pathway Ca ATPase (SPCA1)

Plasma membrane Ca²⁺-ATPase 2 (PMCA2) knockout mice showed that ∼60% of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca²⁺-ATPase’s 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca²⁺/H⁺ antiporter (TMEM165) is expressed sufficiently in the Golgi of lactating mammary tissue to be a relevant contributor to secretory pathway mammary calcium transport. TMEM165 shows marked expression on day one of lactation when compared to timepoints prepartum. At peak lactation TMEM165 expression was 25 times greater than that of early pregnancy. Forced cessation of lactation resulted in a rapid ∼50% decline in TMEM165 expression at 24 h of involution and TMEM165 expression declined 95% at 96 h involution. It is clear that the timing, magnitude of TMEM165 expression and its Golgi location supports a role for this Golgi Ca2⁺/H⁺ antiporter as a contributor to mammary Golgi calcium transport needs, in addition to the better-characterized roles of SPCA1&2.     

The expression, activity and localisation of the secretory pathway Ca-ATPase (SPCA1) in different mammalian tissues

The distribution of the secretory pathway Ca²⁺-ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney.SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca²⁺ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca²⁺ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.     

The pancreas-specific form of secretory pathway calcium ATPase 2 regulates multiple pathways involved in calcium homeostasis

Acinar cell exocytosis requires spatiotemporal Ca²⁺ signals regulated through endoplasmic reticulum (ER) stores, Ca²⁺ATPases, and store-operated Ca²⁺ entry (SOCE). The secretory pathway Ca²⁺ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca²⁺ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca²⁺ homeostasis in a similar fashion to the full-length SPCA2.Using epitope-tagged SPCA2C (SPCA2C^FLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca²⁺] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca²⁺], Ca²⁺ release in response to carbachol, ER Ca²⁺ stores, and store-mediated and independent Ca²⁺ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca²⁺ entry were independent of Orai1. These findings indicate SPCA2C influences Ca²⁺ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca²⁺ regulation.     

Membranes of mammary gland : X. Adenosine triphosphate dependent calcium accumulation by golgi apparatus rich fractions from bovine mammary gland

Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg²⁺-dependent, Ca²⁺-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca²⁺ in vitro. Accumulation of Ca²⁺ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca²⁺ and moderately stimulated by high concentrations of K⁺. This activity was unaffected by Na⁺ and was not inhibited by ouabain. The pH optimum for the Mg²⁺-dependent hydrolysis of ATP was 7.5, the K_m was 5 × 10⁻⁵ M and the activation energy was 6 000 calories/mole. This Mg²⁺-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca²⁺. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca²⁺ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.     

Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

Mammary gland involution is associated with rapid down regulation of major mammary Ca-ATPases

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca²⁺-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca²⁺-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca²⁺-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca²⁺-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca²⁺-ATPases, required by the mammary gland to regulate the large amount of calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca²⁺ overload, calcium mediated cell death and thus play a part in early signaling of mammary involution.     

Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network

Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.     

Ca(2+) signalling in the Golgi apparatus

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments.The Golgi apparatus has been also shown to be involved in Ca²⁺ signalling: it is indeed endowed with Ca²⁺ pumps, Ca²⁺ release channels and Ca²⁺ binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca²⁺ signal within the cell, though this role is still poorly understood.Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca²⁺ handling and selective reduction of Ca²⁺ concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology.In this paper we review the available information on the Ca²⁺ toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.     

Vesicular Calcium Regulates Coat Retention,Fusogenicity, and Size of Pre-Golgi Intermediates

The significance and extent of Ca²⁺ regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca²⁺ with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca²⁺ in the early secretory pathway. Specific depletion of luminal Ca²⁺ in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca²⁺ in COPII vesicle homotypic fusion was demonstrated in vitro using Ca²⁺ chelators. The EF-hand–containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca²⁺-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca²⁺ on homotypic fusion. Immunoisolation established that Ca²⁺ chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca²⁺, acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca²⁺-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.