TICB-1056; No. of Pages 10

Review

Secretory cargo sorting at the trans-Golgi network Christine Kienzle and Julia von Blume Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

Sorting of proteins for secretion from cells is crucial for normal physiology and the regulation of key cellular events. Although the sorting of lysosomal hydrolases at the trans-Golgi network (TGN) for delivery to prelysosomes is well characterized, the corresponding mechanism by which secreted proteins are sorted for plasmamembrane delivery remains poorly understood. Recent discoveries have revealed a novel sorting mechanism that requires the linkage between the cytoplasmic actin cytoskeleton to the membrane-anchored Ca2+ ATPase, SPCA1 (secretory pathway calcium ATPase 1), and the luminal 45 kDa Ca2+-binding protein, Cab45, for successful sorting of a subset of proteins at the TGN. We review progress in understanding these processes. Protein secretion at a glance Protein secretion is crucial for cell–cell communication and for the establishment and maintenance of tissue integrity. Secreted proteins include signaling molecules such as hormones, cytokines, and chemokines, as well as extracellular matrix proteins such as collagens, which provide structural and biochemical support to the surrounding cells and play a major role in cell adhesion, cell–cell communication, and differentiation. Defects in the release of these proteins underlie disorders including autoimmune diseases [1], cancer [2], neurological diseases [3], diabetes [4], and skeletal dysplasia [5]. The majority of studies have focused on the downstream properties of these proteins, but little is known about the mechanism controlling their export from the Golgi apparatus. Most secreted proteins contain a signal sequence that targets them to the endoplasmic reticulum (ER) during synthesis [6]. There, they are translocated into the ER lumen and, once folded, travel in coat protein complex II (COPII)-coated vesicles to the Golgi apparatus. After passage through the Golgi membranes, proteins are sorted at the TGN for transport to their respective cellular compartments or for secretion by specific transport carriers [7–9]. Depending on the cell type, these destinations include apical and basolateral cell surfaces, early/ sorting endosomes, late endosomes, recycling endosomes, secretory storage granules, preceding Golgi compartments, and the ER [10,11] (Figure 1), indicating that Corresponding author: von Blume, J. ([email protected]). Keywords: Ca2+; TGN; protein sorting; secretory cargo. 0962-8924/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tcb.2014.04.007

for each destination at least one distinct type of vesicle might be used [12]. We summarize here our current knowledge regarding the sorting of soluble secretory proteins at the TGN and highlight a novel, receptor-independent mechanism. TGN organization and cargo export The TGN generates a large number of distinct carriers that specialize in the transfer of cargo to various cellular compartments and to the cell surface for secretion. Indeed, the vesicles identified to bud from the TGN are clathrin-coated vesicles moving towards the pre-lysosomes [12], and vesicles destined for the apical and basolateral membrane domains in polarized and non-polarized cells [13] and secretory granules [14]. An important challenge for the field is to understand how the distribution of these molecules is achieved and linked to the organization of the TGN. The TGN structure and size varies among cell types [15]. In most cells the TGN represents a tubular network that emerges from the last two trans cisternae [10]. In contrast to export from the ER, which occurs at stable export domains termed ER exit sites [16], export from the TGN appears to be more complicated. High-voltage electron microscopy combined with computer axial tomography revealed the existence of different domains of the TGN [17], termed ‘exit domains’, which are composed of varying types of vesicular and tubular carriers enriched in cargo and budding machineries but are devoid of Golgi-resident proteins [18]. Indeed, clathrin-coated vesicles that facilitate transport of mannose-6-phosphate receptors (MPR) to endosomes appear to emanate from the last TGN cisternae and its originating tubules. Furthermore, in normal rat kidney epithelial (NRK) cells, the penultimate two cisternae are decorated with a ‘lace-like’ coat that was suggested to represent the export domain for vesicles targeted to the cell surface [17,19]. In particular, epithelial cells that line the surfaces of organs show a polarized distribution of proteins and lipids at their apical and basolateral cell surfaces. Interestingly, this polarized separation also occurs in non-polarized cells [20,21], indicating that similar pathways occur in both cell types. Visualization of carriers destined for the cell surface in living cells revealed structures of distinct and heterogeneous sizes and shapes [22,23]. However, it was unclear whether overexpressed cargo proteins used to detect these structures influenced the observed structures. A new class of transport carriers was recently identified, named CARTS (carriers from the trans-Golgi to the cell surface), providing a Trends in Cell Biology xx (2014) 1–10

1

TICB-1056; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Lysosome Endosome

Apical transport vesicles

Secretory storage granules Basolateral transport vesicles

TGN

trans Medial

COPI

Golgi complex

cis

COPI ERGIC

COPII

Endoplasmic reculum TRENDS in Cell Biology

Figure 1. Protein trafficking and sorting in the secretory pathway. Proteins containing a signal sequence are translocated across the endoplasmic reticulum (ER) membrane. After folding in the ER, proteins have two fates: either they become ER-resident proteins or leave the ER in coat protein complex II (COPII)-coated vesicles towards the Golgi apparatus via the ER Golgi intermediate compartment (ERGIC). After traversing the cis and medial Golgi compartments, proteins enter the trans-Golgi network (TGN), an important sorting station. Here proteins have to be sorted and packaged into transport carriers to reach their final destinations. Numerous TGN exit routes have been described: proteins packaged into clathrin-coated vesicles are transported to the endosomes, whereas constitutively secreted cargo or transmembrane proteins are transported towards the apical or basolateral plasma membrane. In specialized cells, proteins leave the TGN to be stored in secretory storage granules. These granules fuse with the plasma membrane primarily in response to an extracellular stimulus. Retrieval of ER proteins is carried out by COPI vesicles.

new understanding of the composition of carriers [24]. CARTS transport PAUF (pancreatic adenocarcinoma upregulated factor) and LysC (lysozyme C), but not collagen I and vesicular stomatitis virus glycoprotein (VSV-G), from the Golgi to the cell surface (Figure 2). This finding provided clear evidence that cargo molecules are sorted at, and exit from, particular TGN domains created by unique lipid protein microenvironments for which particular cargo molecules have a selective affinity. These domains are composed 2

of lipids such as phosphoinositides, sphingolipids, and cholesterol [25], and/or are decorated with particular coats, tethers, and GTPases required for cargo sorting and vesicle generation [18]. The formation of these export domains is likely highly dynamic and may be dependent on cargo influx. Furthermore, some of these domains may be consumed as export carriers [10]. Although it is assumed that there are more classes of vesicles originating from the TGN, the challenge for

TICB-1056; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Box 1. Principles of sorting of transmembrane proteins at the TGN During transmembrane protein sorting, adaptor proteins (APs) are recruited to the TGN membrane by Arf GTPases. APs then recognize sorting signals in the cytosolic domains of transmembrane proteins. At the TGN, for instance AP-1 attaches clathrin to the membrane and recruits accessory proteins that regulate vesicle formation [86]. In polarized cells it was shown that the clathrin adaptor protein 1-A (AP1A), which is ubiquitously expressed [87], localizes to phosphatidyl inositol 4-phosphate (PI4P) and Arf-1 enriched TGN domains [88,89], as well as to early endosomes, and is required for endosomal targeting. Proteins that travel to the basolateral cell surface in an AP1B-dependent manner [87,90] first move from the TGN into recycling endosomes in a Rab13-dependent manner before they are transported to the basolateral membrane [91,92]. However, it was also suggested that AP-1A and AP1-B localize indistinguishably to the TGN and recycling endosomes, and only differ in their selectivity/affinity for particular cargo motifs [93]. Indeed, endosomal and some basolateral-directed proteins contain tyrosine-based residues (NPXY or YXXF motifs) and domains that contain two leucine residues [(DE)XXL(LI)] or DXXLL consensus motifs that bind to AP-1 [94,95]. It

researchers is to identify markers of these vesicles that allow their characterization and possible purification. Moreover, it remains to be determined if there are different exit domains for distinct classes of secreted proteins. TGN sorting of soluble proteins The TGN is responsible for the sorting of secretory as well as transmembrane cargo or proteins attached to membranes, for example, by glycosyl phosphatidyl inositol (GPI) anchors. Currently several models have been proposed for how transmembrane proteins are sorted at the TGN (Box 1), and for details we refer to excellent reviews and research articles [13,26–28]. Instead, the focus of this review is on the sorting of secretory proteins. Sorting by selective aggregation Professional secretory cells such as neuroendocrine cells synthesize and process peptide hormones that are stored in secretory storage granules. These granules fuse with the plasma membrane and release their contents into the extracellular space in response to an extracellular stimulus [14]. The sorting of these regulated cargoes at the TGN has been the topic of intensive investigation over the past 30 years, and details can be found elsewhere [29–32]. A variety of proteins destined to be sorted into secretory storage granules form large complexes and aggregates within the lumen of the TGN; the low pH (6.4) of this compartment favors the formation of these clusters, which segregate them from other soluble proteins in the TGN. So-called immature secretory granules (ISG) bud from the TGN and transform into mature secretory granules (MSG) that contain a proteinaceous dense core (Figure 2). Mis-sorted non-granule proteins are progressively withdrawn by clathrin-coated vesicles, eventually leaving the correct cargo protein in the MSGs [33]. The ability of regulated secretory cargo, such as granins, to form aggregates is often an intrinsic feature of their protein structures. For example, they contain numerous acidic amino acids that drive clustering in the presence of millimolar Ca2+ and the slightly acidic pH of the TGN [34]. These granin complexes interact directly or indirectly with cholesterol- and sphingolipid-rich

has also been suggested that AP-1 together with the Arf-related protein Arfrp1/Arl-3 is required for the export of the planar cell polarity signaling molecule Vangl2 from the TGN to the plasma membrane through a YYXXF motif [96]. Another adaptor protein at the TGN is AP-4, which binds the [YX(FYL)(FL)E] motif. In contrast to other APs, AP-4 does not assemble clathrin coats but seems to be required for the sorting of a subset of neuronal cargoes [28]. The sorting of proteins destined to the apical cell surface was suggested to occur in lipid raft domains, which are dynamic, nanometer-sized, sterol/sphingolipid-enriched lipid–protein assemblies [97] that can be induced to form stable lipid platforms at the TGN [98]. In addition, the length and the asymmetric amino acid composition of integral membrane proteins, and consequently the lipid bilayer thickness, determine the Golgi retention of transmembrane proteins [99]. In yeast, the exomer, a coat protein complex, is required for the transport of chitin synthase Chs3p and Fus1p from the late Golgi membrane to the plasma membrane [100,101]. However, there are no known orthologs of exomer in higher eukaryotes.

luminal membrane domains in the TGN. This interaction provides a driving force to induce budding from the TGN membranes to form ISGs [35]. It has also been proposed that apical sorting is based on the aggregation of N-linked and O-linked glycans of proteins and lipids. These aggregates are then bound by lectins such as galectin-3 that act as sorting receptors and direct glycoproteins into apical transport vesicles [36]. Receptor-mediated luminal protein sorting at the TGN The sorting of proteins to secretory storage granules was suggested also to occur in a sorting receptor-dependent manner. It was proposed that carboxypeptidase E (CPE) [37] is sorted to secretory storage granules by the interaction of a short a-helical domain with the granule membrane [32]. Membrane-‘tethered’ CPE has been suggested to interact with several cargo proteins including proenkephalin, proinsulin, proopiomelanocortin (POMC) [38], and brain-derived neurotrophic factor (proBDNF) [39] for secretory storage granule targeting. It was also reported that secretogranin III (SGIII) interacts with cholesterol-rich membrane domains in the TGN and binds to chromogranin A (CgA) and other prohormones, and together with CPE facilitates the retention in maturing granules [40,41]. However, the role of CPE as a cargo receptor for proinsulin and POMC has been questioned because proinsulin and POMC are targeted correctly to secretory storage granules in the fat/fat mouse which harbors a mutation in the Cpe/ CPE gene that causes an endocrine disorder [42]. The sorting of lysosomal hydrolases is well understood. This class of proteins is composed of approximately 50 acid hydrolases, including glycosidases, proteases, lipases, nucleases, phosphatases, and sulfatases, which are sorted at the TGN for transport to pre-lysosomes [43]. The oligosaccharide chains of the lysosomal hydrolases acquire terminal mannose-6-phosphate (M6P) residues as they pass through the Golgi complex [44]. These M6P tags bind to MPRs, which contain cytosolic sorting motifs for packaging into transport vesicles. Specifically, MPRs contain ‘acidic cluster dileucine (AC-LL) signals’ that are recognized by GGA (Golgi-localizing, g-adaptin ear domain 3

TICB-1056; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Lysosome Endosome

?

Key:

M6PR Clathrin coat

MSG

M6P tag Lysosomal hydrolases Protein aggregaon Sorlin

ISG

Apical/basolateral soluble cargo (A)

CARTS (B) (C)

Ca2+ ↑pH pH↓

PAUF Lysozyme C

(D)

?

TGN TRENDS in Cell Biology

Figure 2. Sorting of secretory proteins at the TGN is poorly understood. There are two well-described sorting principles at the TGN. This is the (A) recepter-mediated sorting of lysosomal hydrolases via M6P (Mannose-6-Phosphate) and the M6P receptor (M6PR) to the lysosome via endosomes, in clathrin-coated vesicles. Another class of soluble proteins is sorted via sortilin and clathrin to endosomes, eventually arriving to lysosomes. A specific signal in these proteins is recognized by sortilin, thereby permitting their sorting. Another process is the sorting of proteins destined to secretory storage granules (B). These molecules aggregate in the presence of low pH and Ca2+ in the TGN and these aggregates condense and mature from immature storage granules (ISG) into mature storage granules (MSG). CARTS transport Lysozyme C and PAUF to the cell surface (C). How other soluble secretory proteins destined to the apical and basolateral cell surface are sorted remains poorly understood (D).

homology, ARF-binding protein) adaptor proteins. Upon binding the AC-LL signal, GGA adapter proteins recruit clathrin from the cytoplasm and a clathrin coat is assembled at the TGN [45]. After coat formation, the vesicles pinch off the TGN and fuse with endosomal intermediates [46] where the low internal pH (pH 5.5) triggers dissociation of lysosomal enzymes from MPRs (Figure 2). Upon release of the cargo, MPRs are transported back to the TGN where they can start a new cycle [47]. Yeast cells utilize a distant ortholog of the MPR termed MRL1 (mannose-6-phosphate receptor-like 1). Although this protein is required for the transport of some acid hydrolases to the vacuole, it is not likely to do so via a M6P interaction [48]. In contrast to the sorting of mammalian lysosomal hydrolases, most yeast acid hydrolases are sorted by an alternative mechanism. Carboxypeptidase Y (CPY) is sorted in the late Golgi by the vacuolar protein sorting 10 (Vps10) cargo receptor [49]. Vps10 is a type I single-pass transmembrane protein that binds to CPY via a sorting motif composed of four amino acids, QRPL [50]; Vps10 transports CPY to a pre-vacuolar, endosomal compartment via clathrin-coated vesicles. In endosomes, CPY dissociates from Vps10, which is then recycled back to the late Golgi. CPY is further processed in endosomes and moves to the vacuole, where it resides in its mature form [51]. Mammalian cells also sort proteins to the lysosome in an MPR-independent manner. Patients with inclusion cell 4

(I cell) disease are unable to add M6P to lysosomal enzyme oligosaccharide chains because the N-acetylglucosamine (GlcNac)-1 phosphotransferase required for M6P modification is mutated, although a subset of lysosomal hydrolases are transported to lysosomes in the cells from these patients [52]. This process appears to be similar to CPY transport in yeast, suggesting that other receptors are responsible for the sorting of lysosomal hydrolases in mammalian cells. Indeed, the hydrolase receptor sphingolipid activator proteins (SAP) cathepsin D and H appear to rely on at least five Vps10 domain-containing proteins: sortilin, SorCS1, SorCS2, SorCS3 and SorLA [53]. Of these, sortilin specifically mediates the transport from the TGN to endosomes (Figure 2). Furthermore, sortilin has been shown to regulate protein sorting from the TGN to specialized secretory vesicles termed mucocysts. The protozoan Tetrahymena thermophile revealed that mucocyst biosynthesis depends on two sorting processes in the TGN. One class of proteins termed Grl (granule lattice) undergo aggregation, whereas a second group of proteins named Grt-1p (granule tip) and Igr-1p (induced upon granule regeneration) do not, and are sorted via a receptor-mediated process, with sortilin as the receptor [54]. This observation suggests that mucocyst biogenesis and lysosome biogenesis may rely on shared machineries. In addition to the above-mentioned receptors, recent evidence also suggest a role for a carrier protein in transporting Wnt (from ‘Wingless/int’) proteins. Wnts are a

TICB-1056; No. of Pages 10

Review conserved family of secreted proteins that act as morphogens to activate diverse pathways required for major developmental processes as well as to maintain adult tissue homeostasis [55]. Although the secretion mechanism of Wnt is poorly understood, it is known that they require the Wnt carrier protein Wntless (Wls) for their transport to the cell surface [56]. It was assumed that Wls acts as a Wnt sorting receptor in the TGN because initial studies showed that epitope-tagged Wls localizes to the Golgi, TGN, endosomes, and the plasma membrane [56]. Interestingly, a study that applied an antibody to detect endogenous untagged Wls showed that a substantial pool of Wls localizes to the ER where it is palmitoylated by porcupine. Palmitoylated Wnts can then be loaded directly onto Wls and be transported from the ER to the plasma membrane [57]. In this respect it is also interesting to note that, in epithelial cells, Wls together with Wnt3a are sorted from the TGN to the basolateral surface in an activator protein 1 (AP-1)- and clathrin-dependent manner [58]. Therefore, it may be possible that Wls acts as a sorting receptor at the ER and the TGN. The mechanism of Wls acting as sorting receptor in two different compartments remains to be determined. ADF/cofilin-, actin-, Ca2+- and Cab45-dependent cargo sorting A genome-wide screen revealed a requirement for the actin-severing protein twinstar, for secretion of signal sequence-bearing horseradish peroxidase (ss-HRP) from Drosophila S2 cells [59]. Further examination of the process showed that twinstar and its orthologs, in both yeast (Cof1) and mammalian cells (ADF and cofilin 1), facilitate sorting of a subclass of cargo molecules at the TGN. This observation was surprising because twinstar and its orthologs are all cytosolic proteins with no known association with the TGN. A subsequent study using a TGN-localized Ca2+ sensor showed that ADF/cofilin transiently localizes to the TGN membranes and regulates Ca2+ influx into the TGN by interacting with SPCA1 [60]. Secretory proteins sorted in a ADF/cofilin-, actin-, and SPCA1-dependent manner include Ca2+-binding proteins such as cartilage oligomeric protein (COMP), matrix glia protein (MGP), thrombospondin3 (TSP3), and proteins that do not bind Ca2+ such as the metalloproteinase inhibitor TIMP1 (tissue inhibitor of metalloprotease 1) and LysC [61,62]. The process seems to be conserved because Cof1 and Pmr1 (plasma membrane ATPase related), the yeast ortholog of SPCA1, are required for cargo sorting of a subset of secreted proteins from the late Golgi [63], and mutations in Pmr1 affect the sorting of CPY and prehormone maturation in the Golgi membranes [64]. These findings suggested a novel, conserved mechanism of secretory cargo sorting at the TGN. SPCA1 is a TGN-resident transmembrane protein, encoded by the ATP2C1 (ATPase, Ca2+ transporting, type 2C, member 1) gene, that pumps Ca2+, as well as Mn2+, into the lumen of the TGN in an ATP-dependent manner [65]. Loss of one copy of the ATP2C1 gene causes Hailey–Hailey disease, an autosomal dominant skin disorder characterized by suprabasal acantholysis of keratinocytes [66]. Mouse Atp2c1 null mutants do not survive beyond gestational day 10.5 owing to an impairment of neural tube

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

closure. The ultrastructure of these embryos shows an aberrant Golgi morphology with dilated and fewer cisternae as well as increased vesicles [67]. SPCA1 is also required for neuronal differentiation. In N2a mouse neuroblastoma cell lines and hippocampal neurons depleted of SPCA1, polarized trafficking is disrupted, resulting in impairment of neuronal differentiation as well as the generation of functional neurites [68]. Furthermore, SPCA1 RNA interference (RNAi) impairs the trafficking of amyloid precursor protein (APP) and the transport of the purinergic receptor P2X7 [69]. Taken together, these studies show that SPCA1 is required for the transport of physiologically relevant proteins. The concentration of Ca2+ in the Golgi apparatus is heterogeneous, and it has been suggested that there is a Ca2+ gradient across the secretory pathway, from the ER to the TGN [70]. The use of a TGN-specific Ca2+ sensor revealed that luminal Ca2+ content is low and solely relies on the activity of SPCA1 for Ca2+ uptake [69,70]. Nonetheless, how can actin and ADF/cofilin influence the pumping activity of SPCA1 and how does luminal Ca2+ mediate protein sorting? The actin cytoskeleton is known to organize membrane-associated proteins into particular functional domains at the plasma membrane [71]. Its attachment to the membrane can affect phase-separation of initially homogeneous lipids in the membrane and stabilize the phase-separated domains [72]. Therefore, actin and ADF/cofilin could act in a similar manner at the TGN. One possibility is that ADF/cofilin and actin may concentrate SPCA1 molecules into a sorting domain, and this clustering may be necessary for local Ca2+ accumulation within the TGN. Subsequently, cargoes that have a high affinity for Ca2+ would be sequestered into the sorting domain, followed by segregation of the domain (by membrane fission) to generate a cargo-filled transport carrier for transport to the cell surface [60]. It was shown that SPCA1 accumulates in cholesterol-rich membrane domains, and depletion of cholesterol results in a 50% reduction in pumping activity [73]. Collection of SPCA1 into a cholesterol-rich domain stabilized by actin at the TGN may be important for its regulation. It is also possible that the actin cytoskeleton directly regulates the function of the pump via differential binding of monomers/oligomers versus filaments. According to this mechanism, the polymerization state of actin may modulate ion transport. Alternatively, the pump–actin interaction may indirectly affect the pump function. Analogously, the recruitment of cofilin 1 and actin near SPCA1 may alter the local ionic cytoplasmic environment because cofilin 1 binding to filaments is coupled to ion release [74] owing to the opening of a specific ‘stiffness’ site along the filament [75]. The cytoplasmic increase in ions would activate ion transport into the TGN, supporting cargo sorting. How could Ca2+ in the TGN lumen facilitate cargo sorting? Interestingly, cells that are depleted of ADF/cofilin or SPCA1 do not only mis-sort secreted proteins but also release the soluble Golgi-resident protein, Cab45 [60,61]. Lodish and colleagues identified Cab45 in 1996 [76] as a Golgi-resident protein with 6 EF hand (from ‘glutamate/ phenylalanine’) domains that each bind Ca2+. Localization of Cab45 is sensitive to Ca2+ levels within the Golgi 5

TICB-1056; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Key:

(A)

Cofilin1 inacve Ca2+ ions Cab45 Secretory cargo Acn filaments

Cytosol TGN membrane TGN lumen Low Ca2+ pumping acvity

Key:

(B)

Cofilin1 acve Cab45 + cargo

Cytosol TGN membrane TGN lumen High Ca2+ pumping acvity

(C)

Budding vesicle

TGN membrane Dissociaon

?

High Ca2+ pumping acvity

TGN lumen TRENDS in Cell Biology

Figure 3. A novel sorting pathway independent of a ‘classical sorting receptor’. (A) When cofilin 1 is inactive and does not localize to the TGN, actin filaments do not connect to SPCA1 and the pump has a low Ca2+-pumping activity. (B) As soon as cofilin 1 is activated, it binds to SPCA1 and actin filaments are recruited to the trans-Golgi network (TGN), consequently activating the pump by an unknown mechanism and inducing an influx of Ca2+ ions into a specific domain of the TGN. Luminal Ca2+ binds to Cab45 (45 kDa Ca2+-binding protein), and this probably induces a conformational change that allows Cab45 binding to secreted proteins. Binding is proposed to segregate these proteins from other proteins present in the TGN. (C) To be packaged into a transport carrier, the secreted proteins must dissociate from Cab45. How this occurs is not known, but could involve a post-translational modification such as phosphorylation. How Cab45 is retained in the TGN without accompanying cargo into nascent transport vesicles remains unknown.

6

TICB-1056; No. of Pages 10

Review

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

Box 2. Outstanding questions  How do cofilin 1 and actin mechanistically activate the pump? Localized recruitment of actin filaments via cofilin 1 may retain the pump in distinct TGN domains in which the pump is active. Alternatively, the pump activation could rely on oligomerization of the protein, which has been shown for several P-type ATPases such as the Na+/K+ ATPase [102] and the H+ ATPase [103]. The oligomerization status may be supported by the actin cytoskeleton that stabilizes a membrane microdomain.  How can Cab45 sort proteins into a vesicle without also being secreted? If Cab45 binds to secreted proteins, they will need to dissociate from Cab45 before packaging into a transport carrier. This process could be mediated by a decrease in local Ca2+ levels or by a post-translational modification such as phosphorylation. Future work is needed to clarify these essential events, which are crucial for protein sorting at the TGN. It will be necessary to identify novel major players in the pathway by pull-down analysis and mass spectrometry. The process and the role of each player should be

because disruption of the Ca2+ concentration by ionophore treatment leads to Cab45 secretion from cells [62]. Cab45 itself may be required for SPCA1-dependent Ca2+ influx into the TGN because preliminary studies suggest it may colocalize with SPCA1 in a particular TGN-positive region (C.K. and J.v.B., unpublished). Therefore, it is possible that Cab45 modulates the pump activity internally via a feedback mechanism. This hypothesis needs to be tested by TGN-targeted Ca2+ sensors and super-resolution microscopy to determine if Ca2+ influx is restricted to a Cab45/ SPCA1-positive domain. In summary, it will be important to determine how the cytosolic complex of cofilin 1 and actin communicate with luminal Cab45. Furthermore, Cab45 binds several secretory proteins including LysC and COMP, and thus appears to facilitate the secretion of these proteins [62]. Cab45 might act as a soluble cargo receptor that requires finely tuned, luminal Ca2+ signals to sequester or release the cargo molecules into transport carriers or into a separate TGN domain. Interestingly, it has been shown that ER-resident Ca2+binding proteins such as GRP78 (78 kDa glucose-regulated protein, also known as binding immunoglobulin protein, BiP) are rapidly secreted from cells when ER Ca2+ levels drop [77], suggesting that Ca2+ levels impair the segregation of ER-resident proteins from secretory proteins, resulting in the transport of cargo molecules to the wrong target membrane [78]. A similar system may exist in the Golgi apparatus. Cab45 could be the Ca2+-binding protein that assembles secreted proteins in a Ca2+–protein matrix that forms when SPCA1 pumps Ca2+ into the lumen of the TGN, thereby sequestering cargo molecules into a specific domain and separating them from other secretory proteins and Golgi residents. In this way, cargo export would occur from a domain in regions where the Ca2+–protein matrix is unable to sustain Ca2+. So far it is not clear how and if the cargo dissociates from Cab45 in Golgi membranes, and this will require further testing. In summary, actin filaments and cofilin 1 bind to SPCA1 at the TGN (Figure 3A), resulting in pump activation, thereby triggering Ca2+ influx into a specific domain of the TGN. This transient, local increase in Ca2+ recruits Cab45, which has a high affinity for Ca2+ and is therefore

characterized in vitro with purified proteins. In addition, dynamic imaging techniques and super-resolution microscopy will be necessary to elucidate the action of actin in modulating SPCA1 in vivo.  Previous work demonstrated that sorting of secretory proteins at the TGN occurs actively. The actin/cofilin 1/SPCA1/Ca2+/Cab45-dependent process may only be among many other sorting activities that occur at the TGN. Future studies should focus on identifying new sorting components and purifying and characterizing TGN to cell surface transport carriers that transport secretory proteins. Novel gene-editing techniques in mammalian cells such as CRISPR/Cas9 [104] coupled to high-throughput screening will allow us to genetically identify more proteins required for sorting and transport of secretory proteins at the TGN. In addition, reconstitution of the components in vitro and super-resolution microscopy will be necessary to elucidate the action of the sorting components at the TGN.

retained within the TGN to bind secretory proteins and segregate them from other TGN protein content (Figure 3B). Subsequent dissociation of the Cab45–cargo complex occurs either upon a decrease in Ca2+ or by a signal such as phosphorylation (Figure 3C), resulting in the segregation of cargo for sorting into a particular class of transport carrier. Indeed, the serine/threonine kinase Fam20C (from ‘family with sequence similarity 20’) was recently identified as the ‘Golgi casein kinase’ which phosphorylates proteins that carry specific S–X–E/pS sites while trafficking through the Golgi apparatus [79]. Fam20C phosphorylates proteins required for biomineralization, and many other secretory proteins contain the substrate sequence [79]. However, the existence and the function of these modifications remain to be elucidated. It may be that phosphorylation plays a major role in cargo– Cab45 interaction and dissociation. Concluding remarks Several studies indicate that constitutive secretion involves bulk transport through the secretory pathway unless the protein contains a positive sorting signal for diversion to lysosomes or secretory storage granules [80,81]. Therefore, it was postulated that secretory proteins are not actively sorted and their packaging into vesicles at the TGN occurs by default [82]. In the past three decades enormous strides have been made in identifying the sorting signals and molecular requirements of different TGN exit pathways. Protein segregation is now established as playing an essential role in sorting proteins into secretory storage granules [30], and signals have been identified that are responsible for the sorting of acid hydrolases to pre-lysosomes [83]. However, recent screens have uncovered unexpected roles for actin, cofilin 1, and calcium signals for the process by which proteins are constitutively secreted from cells (Figure 2). Furthermore, a recent study provided evidence that secretory proteins are actively sorted at the TGN and that constitutive cargo use distinct carriers for transport to the cell surface. How are secreted proteins sorted? The simplest concept invokes a ligand–receptor interaction where a sorting signal on the cargo binds to a receptor in the TGN. In the early Golgi, the KDEL receptor recognizes the KDEL 7

TICB-1056; No. of Pages 10

Review (Lys–Asp–Glu–Leu) sequence of escaped, ER-resident proteins and returns them to the ER in COPI-coated vesicles (Figure 1) [84,85]. However, a receptor for secreted proteins at the TGN has not been identified. Therefore, sorting of secreted proteins may utilize an alternative mechanism. For particular secretory cargoes, sorted independently of a classical receptor [60], sorting depends on a transient influx of Ca2+ into the TGN lumen. This increase in luminal Ca2+ is induced by the binding of actin and cofilin 1 to SPCA1 on the cytosolic face of the TGN, and it facilitates the association of the secretory proteins with the Golgiresident protein, Cab45. Cab45 might act as a ‘soluble receptor’ [62] to segregate a subset of secretory proteins. The process appears to be conserved in evolution because twinstar was identified in Drosophila, and mammalian cells and yeast require the same components (actin, cofilin, Pmr1, Ca2+) for sorting of soluble proteins. However, a yeast ortholog of Cab45 is currently unknown. Much remains to be learned regarding this sorting process, and future studies should be directed at answering the remaining outstanding questions (Box 2). Acknowledgments We thank Josse van Galen for insightful discussions and proofreading of the manuscript. The group of J.v.B. is funded by an Emmy Noether fellowship (project BL 1186/1-1) from the Deutsche Forschungsgemeinschaft (DFG) and by a European Commission Framework Program (FP7) Marie Curie Career Reintegration grant.

References 1 Antonelli, A. et al. (2011) Circulating chemokine (CXC motif) ligand (CXCL)9 is increased in aggressive chronic autoimmune thyroiditis, in association with CXCL10. Cytokine 55, 288–293 2 Welsh, J.B. et al. (2003) Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum. Proc. Natl. Acad. Sci. U.S.A. 100, 3410–3415 3 Chen, C.D. et al. (2001) Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca2+ stabilization. EMBO J. 20, 6277– 6287 4 Harding, H.P. et al. (2001) Diabetes mellitus and exocrine pancreatic dysfunction in Perk / mice reveals a role for translational control in secretory cell survival. Mol. Cell 7, 1153–1163 5 Maddox, B.K. et al. (1997) The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia. J. Biol. Chem. 272, 30993–30997 6 Blobel, G. (1980) Intracellular protein topogenesis. Proc. Natl. Acad. Sci. U.S.A. 77, 1496–1500 7 Griffiths, G. and Simons, K. (1986) The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234, 438–443 8 Glick, B.S. and Nakano, A. (2009) Membrane traffic within the Golgi apparatus. Annu. Rev. Cell Dev. Biol. 25, 113–132 9 Campelo, F. and Malhotra, V. (2012) Membrane fission: the biogenesis of transport carriers. Annu. Rev. Biochem. 81, 407–427 10 De Matteis, M.A. and Luini, A. (2008) Exiting the Golgi complex. Nat. Rev. Mol. Cell Biol. 9, 273–284 11 Pfeffer, S.R. (2011) Entry at the trans-face of the Golgi. Cold Spring Harb. Perspect. Biol. 3, a005272 12 Traub, L.M. and Kornfeld, S. (1997) The trans-Golgi network: a late secretory sorting station. Curr. Opin. Cell Biol. 9, 527–533 13 Mellman, I. and Nelson, W.J. (2008) Coordinated protein sorting, targeting and distribution in polarized cells. Nat. Rev. Mol. Cell Biol. 9, 833–845 14 Palade, G. (1975) Intracellular aspects of the process of protein synthesis. Science 189, 867 15 Clermont, Y. et al. (1995) Trans-Golgi network (TGN) of different cell types: three-dimensional structural characteristics and variability. Anat. Rec. (Hoboken) 242, 289–301

8

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

16 Budnik, A. and Stephens, D.J. (2009) ER exit sites – localization and control of COPII vesicle formation. FEBS Lett. 583, 3796–3803 17 Ladinsky, M.S. et al. (1994) HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat. J. Cell Biol. 127, 29–38 18 Gleeson, P.A. et al. (2004) Domains of the TGN: coats, tethers and G proteins. Traffic 5, 315–326 19 Pfeffer, S. (2003) Membrane domains in the secretory and endocytic pathways. Cell 112, 507–517 20 Mu¨sch, A. et al. (1996) Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells. J. Cell Biol. 133, 543–558 21 Yoshimori, T. et al. (1996) Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J. Cell Biol. 133, 247–256 22 Toomre, D. et al. (1999) Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells. J. Cell Sci. 112, 21–33 23 Polishchuk, E.V. et al. (2003) Mechanism of constitutive export from the Golgi: bulk flow via the formation, protrusion, and en bloc cleavage of large trans-Golgi network tubular domains. Mol. Biol. Cell 14, 4470–4485 24 Wakana, Y. et al. (2012) A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. EMBO J. http://dx.doi.org/10.1038/emboj.2012.235 25 Holthuis, J.C.M. and Levine, T.P. (2005) Lipid traffic: floppy drives and a superhighway. Nat. Rev. Mol. Cell Biol. 6, 209–220 26 Burgos, P.V. et al. (2010) Sorting of the Alzheimer’s disease amyloid precursor protein mediated by the AP-4 complex. Dev. Cell 18, 425–436 27 Anitei, M. and Hoflack, B. (2011) Exit from the trans-Golgi network: from molecules to mechanisms. Curr. Opin. Cell Biol. 23, 443–451 28 Bonifacino, J.S. (2014) Adaptor proteins involved in polarized sorting. J. Cell Biol. 204, 7–17 29 Tooze, S.A. and Huttner, W.B. (1990) Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell 60, 837–847 30 Chanat, E. and Huttner, W.B. (1991) Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J. Cell Biol. 115, 1505–1519 31 Borgonovo, B. et al. (2006) Biogenesis of secretory granules. Curr. Opin. Cell Biol. 18, 365–370 32 Dikeakos, J.D. and Reudelhuber, T.L. (2007) Sending proteins to dense core secretory granules: still a lot to sort out. J. Cell Biol. 177, 191–196 33 Arvan, P. and Castle, D. (1998) Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem. J. 332, 593–610 34 Gerdes, H.H. and Glombik, M.M. (1999) Signal-mediated sorting to the regulated pathway of protein secretion. Ann. Anat. 181, 447–453 35 Kim, T. et al. (2005) Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis. J. Neurosci. 25, 6958–6961 36 Delacour, D. et al. (2006) Requirement for galectin-3 in apical protein sorting. Curr. Biol. 16, 408–414 37 Dhanvantari, S. et al. (2002) Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion. Biochemistry 41, 52–60 38 Cool, D.R. and Loh, Y.P. (1998) Carboxypeptidase E is a sorting receptor for prohormones: binding and kinetic studies. Mol. Cell. Endocrinol. 139, 7–13 39 Lou, H. et al. (2005) Sorting and activity-dependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase E. Neuron 45, 245–255 40 Hosaka, M. et al. (2004) Secretogranin III binds to cholesterol in the secretory granule membrane as an adapter for chromogranin A. J. Biol. Chem. 279, 3627–3634 41 Hosaka, M. et al. (2005) Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules. J. Cell Sci. 118, 4785–4795 42 Irminger, J.C. et al. (1997) Proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase E-deficient Cpefat/ Cpefat mice. J. Biol. Chem. 272, 27532–27534

TICB-1056; No. of Pages 10

Review 43 Cooper, G.M. (2000) Lysosomes. In The Cell: A Molecular Approach. (2nd edn), Sinauer Associates (http://www.ncbi.nlm.nih.gov/books/ NBK9839/) 44 Braulke, T. and Bonifacino, J.S. (2009) Sorting of lysosomal proteins. Biochim. Biophys. Acta 1793, 605–614 45 Le Borgne, R. and Hoflack, B. (1997) Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN. J. Cell Biol. 137, 335–345 46 van Meel, E. and Klumperman, J. (2008) Imaging and imagination: understanding the endo-lysosomal system. Histochem. Cell Biol. 129, 253–266 47 Ghosh, P. et al. (2003) Mannose 6-phosphate receptors: new twists in the tale. Nat. Rev. Mol. Cell Biol. 4, 202–212 48 Whyte, J.R. and Munro, S. (2001) A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases. Curr. Biol. 11, 1074–1078 49 Cooper, A.A. and Stevens, T.H. (1996) Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J. Cell Biol. 133, 529–541 50 Valls, L.A. et al. (1990) Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids. J. Cell Biol. 111, 361–368 51 Stack, J.H. et al. (1995) Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTPbinding proteins. Annu. Rev. Cell Dev. Biol. 11, 1–33 52 Glickman, J.N. and Kornfeld, S. (1993) Mannose 6-phosphateindependent targeting of lysosomal enzymes in I-cell disease B lymphoblasts. J. Cell Biol. 123, 99–108 53 Hermey, G. (2009) The Vps10p-domain receptor family. Cell. Mol. Life Sci. 66, 2677–2689 54 Briguglio, J.S. et al. (2013) Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena. J. Cell Biol. 203, 537–550 55 Clevers, H. and Nusse, R. (2012) Wnt/b-catenin signaling and disease. Cell 149, 1192–1205 56 Ba¨nziger, C. et al. (2006) Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522 57 Yu, J. et al. (2014) Wls retrograde transport to the endoplasmic reticulum during Wnt secretion. Dev. Cell 29, 1–15 58 Yamamoto, H. et al. (2013) The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms. J. Cell Sci. 126, 2931–2943 59 Bard, F. et al. (2006) Functional genomics reveals genes involved in protein secretion and Golgi organization. Nature 439, 604–607 60 von Blume, J. et al. (2011) ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1. Dev. Cell 20, 652–662 61 von Blume, J. et al. (2009) Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network. J. Cell Biol. 187, 1055– 1069 62 von Blume, J. et al. (2012) Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network. J. Cell Biol. 199, 1057–1066 63 Curwin, A.J. et al. (2012) Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast. Mol. Biol. Cell 23, 2327–2338 64 Antebi, A. and Fink, G.R. (1992) The yeast Ca2+-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol. Biol. Cell 3, 633–654 65 Missiaen, L. et al. (2007) Calcium in the Golgi apparatus. Cell Calcium 41, 405–416 66 Hu, Z. et al. (2000) Mutations in ATP2C1, encoding a calcium pump, cause Hailey–Hailey disease. Nat. Genet. 24, 61–65 67 Okunade, G.W. et al. (2007) Loss of the Atp2c1 secretory pathway Ca2+-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes. J. Biol. Chem. 282, 26517–26527 68 Sepu´lveda, M.R. et al. (2007) Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in cerebellum relative to other Ca2+ pumps. J. Neurochem. 103, 1009–1018

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

69 Lissandron, V. et al. (2010) Unique characteristics of Ca2+ homeostasis of the trans-Golgi compartment. Proc. Natl. Acad. Sci. U.S.A. 107, 9198–9203 70 Pizzo, P. et al. (2011) Ca2+ signalling in the Golgi apparatus. Cell Calcium 50, 184–192 71 Plowman, S.J. et al. (2005) H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton. Proc. Natl. Acad. Sci. U.S.A. 102, 15500–15505 72 Liu, A.P. and Fletcher, D.A. (2006) Actin polymerization serves as a membrane domain switch in model lipid bilayers. Biophys. J. 91, 4064–4070 73 Baron, S. et al. (2010) The secretory pathway Ca2+-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells. Biochim. Biophys. Acta 1798, 1512–1521 74 Cao, W. et al. (2006) Energetics and kinetics of cooperative cofilin– actin filament interactions. J. Mol. Biol. 361, 257–267 75 Kang, H. et al. (2012) Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness. Proc. Natl. Acad. Sci. U.S.A. 109, 16923–16927 76 Scherer, P.E. et al. (1996) Cab45, a novel Ca2+-binding protein localized to the Golgi lumen. J. Cell Biol. 133, 257–268 77 Booth, C. and Koch, G.L. (1989) Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59, 729–737 78 Sambrook, J.F. (1990) The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum. Cell 61, 197–199 79 Tagliabracci, V.S. et al. (2012) Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science 336, 1150–1153 80 Pfeffer, S.R. and Rothman, J.E. (1987) Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56, 829–852 81 Thor, F. et al. (2009) Bulk flow revisited: transport of a soluble protein in the secretory pathway. Traffic 10, 1819–1830 82 Moore, H.H. and Kelly, R.B. (1986) Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature 321, 443–446 83 Kornfeld, S. and Mellman, I. (1989) The biogenesis of lysosomes. Annu. Rev. Cell Biol. 5, 483–525 84 Munro, S. and Pelham, H.R. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907 85 Pfeffer, S.R. (2007) Unsolved mysteries in membrane traffic. Annu. Rev. Biochem. 76, 629–645 86 Robinson, M.S. and Bonifacino, J.S. (2001) Adaptor-related proteins. Curr. Opin. Cell Biol. 13, 444–453 87 Ohno, H. et al. (1999) Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells. FEBS Lett. 449, 215–220 88 Hirst, J. and Robinson, M.S. (1998) Clathrin and adaptors. Biochim. Biophys. Acta 1404, 173–193 89 Wang, Y.J. et al. (2003) Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114, 299–310 90 Diaz, F. et al. (2009) Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 106, 11143–11148 91 Fo¨lsch, H. et al. (2001) Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. J. Cell Biol. 152, 595–606 92 Nokes, R.L. et al. (2008) Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells. J. Cell Biol. 182, 845–853 93 Guo, X. et al. (2013) The adaptor protein-1 m1B subunit expands the repertoire of basolateral sorting signal recognition in epithelial cells. Dev. Cell 27, 353–366 94 Bonifacino, J.S. and Traub, L.M. (2003) Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447 95 Rodriguez-Boulan, E. and Mu¨sch, A. (2005) Protein sorting in the Golgi complex: shifting paradigms. Biochim. Biophys. Acta 1744, 455–464 96 Guo, Y. et al. (2013) A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network. Elife 2, e00160

9

TICB-1056; No. of Pages 10

Review 97 Lingwood, D. and Simons, K. (2010) Lipid rafts as a membraneorganizing principle. Science 327, 46–50 98 Simons, K. and Gerl, M.J. (2010) Revitalizing membrane rafts: new tools and insights. Nat. Rev. Mol. Cell Biol. 11, 688–699 99 Sharpe, H.J. et al. (2010) A comprehensive comparison of transmembrane domains reveals organelle-specific properties. Cell 142, 158–169 100 Wang, C-W. et al. (2006) Exomer: A coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast. J. Cell Biol. 174, 973–983

10

Trends in Cell Biology xxx xxxx, Vol. xxx, No. x

101 Zanolari, B. et al. (2011) Transport to the plasma membrane is regulated differently early and late in the cell cycle in Saccharomyces cerevisiae. J. Cell Sci. 124, 1055–1066 102 Taniguchi, K. et al. (2001) The oligomeric nature of Na/K-transport ATPase. J. Biochem. 129, 335–342 103 Chadwick, C.C. et al. (1987) A hexameric form of the Neurospora crassa plasma membrane H+-ATPase. Arch. Biochem. Biophys. 252, 348–356 104 Barrangou, R. (2013) CRISPR–Cas systems and RNA-guided interference. Wiley Interdiscip. Rev. RNA 4, 267–278

Secretory cargo sorting at the trans-Golgi network.

Sorting of proteins for secretion from cells is crucial for normal physiology and the regulation of key cellular events. Although the sorting of lysos...
2MB Sizes 2 Downloads 3 Views