Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Commuting between Golgi cisternae--mind the GAP!

Intracellular transport has remained central to cell biology now for more than 40 years. Despite this, we still lack an overall mechanistic framework that describes transport in different parts of the cell. In the secretory pathway, basic questions, such as how biosynthetic cargo traverses the pathway, are still debated. Historically, emphasis was first put on interpreting function from morphology at the ultrastructural level revealing membrane structures such as the transitional ER, vesicular carriers, vesicular tubular clusters, Golgi cisternae, Golgi stacks and the Golgi ribbon. This emphasis on morphology later switched to biochemistry and yeast genetics yielding many of the key molecular players and their associated functions that we know today. More recently, microscopy studies of living cells incorporating biophysics and system analysis has proven useful and is often used to readdress earlier findings, sometimes with surprising outcomes.     

Intermediate organelles of the plant secretory pathway: identity and function

The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.     

Green light for the secretory pathway

Summary Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes.     

Green light for the secretory pathway

Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes.     

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure-function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space.     

A role for kinesin-2 in COPI-dependent recycling between the ER and the Golgi complex

Transport carriers operating between early compartments in the mammalian secretory pathway have to travel long distances in the cell by mostly relying on the microtubule network and its associated motor proteins. Although anterograde transport from the endoplasmic reticulum (ER) to the Golgi complex is mediated by cytoplasmic dynein, the identity of the motor(s) mediating transport in the retrograde direction is presently unclear. Some studies have suggested that the heterotrimeric kinesin-2 complex plays a role in transport between the ER and the Golgi. Here, we have examined kinesin-2 function by using an RNA-interference approach to downregulate the expression of KAP3, the nonmotor subunit of kinesin-2, in HeLa cells. KAP3 silencing results in the fragmentation of the Golgi apparatus and a change in the steady-state localization of the KDEL-receptor (KDEL-R). Using specific transport assays, we show that the rate of anterograde secretory traffic is unaffected in these cells but that KDEL-R-dependent retrograde transport is strongly abrogated. Our data strongly support a role for kinesin-2 in the KDEL-R-/COPI-dependent retrograde transport pathway from the Golgi complex to the ER.     

The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation

After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on “maturing” cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation models predict the progress of such polymerized residents through the stack along with cargo, but stable cisternae models do not. The results support the cisternal maturation mechanism.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

Global approaches to study Golgi function

Enormous insights into Golgi function have been provided by yeast genetics, biochemical assays and immuno-labeling methods and the emerging picture is of a very complex organelle with multiple levels of regulation. Despite many elegant experimental approaches, it remains unclear what mechanisms transport secretory proteins and lipids through the Golgi, and even the basic structure of the organelle is debated. Recently, new, global approaches such as proteomics and functional genomics have been applied to study the Golgi and its matrix. The data produced reveals great complexity and has potential to help address major unresolved questions concerning Golgi function.     

Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.     

Genetic analysis of clathrin function in yeast

The use of yeast mutants to study the function and dynamics of clathrin-coated membranes has offered new insights into clathrin's role in the secretory pathway and has raised additional questions. Most strains of yeast can incur a disruption of clathrin heavy or light chain genes and remain viable. However, in rare cases, alleles of genes other than clathrin affect the viability of clathrin-deficient cells. The relationship of the products of these genes to clathrin awaits clarification. Phenotypic characterization of clathrin-deficient yeast mutants suggests that clathrin is not essential for the generation of secretory pathway transport vesicles at the ER or the Golgi complex but is required for the intracellular retention of a Golgi membrane protein, Kex2p. With this genetic evidence for clathrin's function in vivo, biochemical and genetic experiments can be designed to address the mechanism by which clathrin effects retention of Kex2p. Clathrin-deficient yeast carry out protein secretion, receptor-mediated endocytosis of mating pheromone, and efficient targeting of newly synthesized vacuolar proteins. These observations challenge aspects of clathrin's proposed involvement in protein transport through the secretory pathway and to lysosomes in mammalian cells. However, the differences are beginning to recede in the face of additional experiments; the formation of clathrin coated vesicles is no longer commonly thought to be obligately coupled to transport through the secretory pathway in mammalian cells (Rothman 1986; Brodsky, 1988), and the role of clathrin in retaining a Golgi membrane protein in yeast may have its precedents in receptor-mediated endocytosis by mammalian cells or in secretory granule formation in endocrine cells. A unified theory of clathrin function is emerging (Brodsky, 1988) which suggests that the clathrin coat assemblage (clathrin heavy and light chains and the associated proteins) acts as a facilitator of intracellular protein transport by sorting and concentrating cargo molecules. The results from studies of clathrin-deficient yeast support this theory. Future experiments will determine whether clathrin provides its functions at different transport stages in different organisms or whether all eukaryotic cells employ clathrin at the same stages of intracellular protein transport.     

The multiple facets of the Golgi reassembly stacking proteins

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.     

Low density lipoprotein receptor and cation-independent mannose 6- phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells

Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.     

Prohormone transport through the secretory pathway of neuroendocrine cells.

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.     

Regulation of traffic and organelle architecture of the ER-Golgi interface by signal transduction

The components that control trafficking between organelles of the secretory pathway as well as their architecture were uncovered to a reasonable extent in the past decades. However, only recently did we begin to explore the regulation of the secretory pathway by cellular signaling. In the current review, we focus on trafficking between the endoplasmic reticulum and the Golgi apparatus. We highlight recent advances that have been made toward a better understanding of how the secretory pathway is regulated by signaling and discuss how this knowledge is important to obtain an integrative view of secretion in the context of other homeostatic processes such as growth and proliferation.     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

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.