BBA - Molecular Cell Research (v.1744, #3)

The Golgi complex by Rainer Duden; John Presley; Brian Storrie (257-258).

Classically, the secretory pathway has been studied using a combination of electron microscopic, biochemical and genetic approaches. In the last 20 years with the arrival of molecular biology and epitope tagging, fluorescence microscopy has become more important than previously. Moreover, with the common availability of Green Fluorescent Protein (GFP) and confocal microscopes in the last 10 years, live cell imaging has become a major experimental approach. This review highlights the impact of the recent introduction of single-cell quantitative time-lapse imaging and photobleach techniques on the study of the secretory pathway, and the potential impact of those optical techniques which may play a significant future role in the study of the Golgi apparatus and the secretory pathway. Particular attention is paid to techniques (Fluorescence Resonance Energy Transfer, Fluorescence Correlation Spectroscopy) which can monitor protein–protein interactions in living cells.
Keywords: Golgi apparatus; Green fluorescent protein; Microscopy; Fluorescence;

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.
Keywords: Mammalian; Golgi; EM; Tomography; Membrane traffic;

COPII and exit from the endoplasmic reticulum by Bor Luen Tang; Ya Wang; Yan Shan Ong; Wanjin Hong (293-303).
First discovered by genetic analysis of yeast secretion mutants, the evolutionarily conserved vesicular coat protein II (COPII) complex is responsible for membrane transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In recent years, extensive efforts in structural, morphological, genetic and molecular analysis have greatly enhanced our understanding of the structural and molecular basis of COPII subunit assembly and selective cargo packaging during ER export. Very recent data have also indicated that a more “classical” picture of vesicle formation from ER exit sites (ERES) followed by their transport to the Golgi is far from accurate. Proteins modulating the function of COPII have also emerged in recent analysis. They either affect COPII-based cargo selection, the formation of vesicle/transport carrier, or subsequent targeting of the transport carrier. Together, elucidation of COPII-mediated ER export has painted a fascinating picture of molecular complexity for an essential process in all eukaryotic cells.
Keywords: Coat protein II; Endoplasmic reticulum; Golgi; Sar1; Sec13/31; Sec24;

The transport of proteins and lipids between the endoplasmic reticulum and Golgi apparatus is initiated by the collection of secretory cargo from within the lumen of the endoplasmic reticulum. Subsequently, transport carriers are formed that bud from this membrane and are transported to, and subsequently merge with, the Golgi. The principle driving force behind the budding process is the multi-subunit coat protein complex, COPII. A considerable amount of information is now available regarding the molecular mechanisms by which COPII components operate together to drive cargo selection and transport carrier formation. In contrast, the precise nature of the transport carriers formed is still a matter of considerable debate. Vesicular and tubular carriers have been characterized that are, or in other cases are not, coated with the COPII complex. Here, we seek to integrate much of the data surrounding this topic and try to understand the mechanisms by which vesicular and/or tubular carriers might be generated.
Keywords: Endoplasmic reticulum; Golgi; COPII; Vesicle; Tubule; Tomography; Live cell imaging; Electron microscopy;

The intimate link between microtubule (MT) organization and the components of the secretory pathway has suggested that MT-based motility is an essential component of vesicular membrane transport and membrane polarization. The molecular details of these processes are still under investigation; however, a novel class of MT plus end-binding proteins shed new light on transport between the endoplasmic reticulum (ER) and Golgi apparatus. The dynactin complex, an initial member of this family, shares localization and live-cell imaging phenotypes with other plus end-binding proteins such as CLIP-170 and EB1. In addition, dynactin has been shown to mediate the binding of ER–Golgi transport vesicles to MTs through a regulated MT-binding motif in p150 Glued . Whereas the plus end-binding activity of CLIP-170 and EB1 has been linked to the regulation of dynamic instability, the plus end binding of dynactin is implicated in a search–capture mechanism for dynein-dependent cargoes. An examination of dynactin's role in ER–Golgi transport suggests that plus end binding could be a reflection of fundamental membrane transport mechanisms.
Keywords: Microtubule; Plus end; Dynamic instability; Dynactin; Cytoplasmic dynein; CLIP-170; Golgi apparatus; Endoplasmic reticulum;

Golgi tethering factors by Vladimir Lupashin; Elizabeth Sztul (325-339).
Transport of cargo to, through and from the Golgi complex is mediated by vesicular carriers and transient tubular connections. In this review, we describe vesicle tethering events with the understanding that similar events occur during transport via larger structures. Tethering factors can be generally divided into a group of coiled-coil proteins and a group of multi-subunit complexes. Current evidence suggests that these factors function in a variety of membrane–membrane tethering events at the Golgi complex, interact with SNARE molecules, and are regulated by small GTPases of the Rab and Arl families.
Keywords: ER; Golgi; Tethering; Protein traffic; Coiled-coil protein;

Intra-Golgi transport: A way to a new paradigm? by Alexander A. Mironov; Galina V. Beznoussenko; Roman S. Polishchuk; Alvar Trucco (340-350).
The morpho-functional principles of intra-Golgi transport are, surprisingly, still not clear, which is in marked contrast to our advanced knowledge of the underlying molecular machineries. Recently, the conceptual and technological hindrances that had delayed progress in this area have been disappearing, and a cluster of powerful morphological techniques has been revealing new glimpses of the organization of traffic in intact cells. Here, we discuss the new concepts around the present models of intra-Golgi transport.
Keywords: Golgi complex; Intracellular transport; Transport models; Molecular mechanisms;

Commuting between Golgi cisternae—Mind the GAP! by Fredrik Kartberg; Markus Elsner; Linda Fröderberg; Lennart Asp; Tommy Nilsson (351-363).
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.
Keywords: Golgi; COPI; Protein transport; Protein sorting; GTPases, ARF1; ARFGAP1, coatomer;

Multiple activities for Arf1 at the Golgi complex by Julie G. Donaldson; Akira Honda; Roberto Weigert (364-373).
The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.
Keywords: Arf1; Golgi complex; COPI; Adaptor protein; Membrane traffic; Phosphoinositide;

Spectrins and the Golgi by Kenneth A. Beck (374-382).
Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.
Keywords: Golgi; Spectrin; Syne-1; Transitional ER; COPII; Nuclear envelope; Neuromuscular junction; Syncytium;

Golgins and GTPases, giving identity and structure to the Golgi apparatus by Benjamin Short; Alexander Haas; Francis A. Barr (383-395).
In this review we will focus on the recent advances in how coiled-coil proteins of the golgin family give identity and structure to the Golgi apparatus in animal cells. A number of recent studies reveal a common theme for the targeting of golgins containing the ARL-binding GRIP domain, and the related ARF-binding GRAB domain. Similarly, other golgins such as the vesicle tethering factor p115 and Bicaudal-D are targeted by the Rab GTPases, Rab1 and Rab6, respectively. Together golgins and their regulatory GTPases form a complex network, commonly known as the Golgi matrix, which organizes Golgi membranes and regulates membrane trafficking.
Keywords: Golgin; Rab; ARF-like; ADP-ribosylation factor; GTPase; Golgi matrix;

The role of the phosphoinositides at the Golgi complex by Maria Antonietta De Matteis; Antonella Di Campli; Anna Godi (396-405).
The phosphorylated derivatives of phosphatidylinositol (PtdIns), known as the polyphosphoinositides (PIs), represent key membrane-localized signals in the regulation of fundamental cell processes, such as membrane traffic and cytoskeleton remodelling. The reversible production of the PIs is catalyzed through the combined activities of a number of specific phosphoinositide phosphatases and kinases that can either act separately or in concert on all the possible combinations of the 3, 4, and 5 positions of the inositol ring. So far, seven distinct PI species have been identified in mammalian cells and named according to their site(s) of phosphorylation: PtdIns 3-phosphate (PI3P); PtdIns 4-phosphate (PI4P); PtdIns 5-phosphate (PI5P); PtdIns 3,4-bisphosphate (PI3,4P2); PtdIns 4,5-bisphosphate (PI4,5P2); PtdIns 3,5-bisphosphate (PI3,5P2); and PtdIns 3,4,5-trisphosphate (PI3,4,5P3). Over the last decade, accumulating evidence has indicated that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated phosphoinositides, but also as regulators of different protein targets in their own right. These regulatory actions are mediated through the direct binding of their protein targets. In this way, the PIs can control the subcellular localization and activation of their various effectors, and thus execute a variety of cellular responses. To exert these functions, the metabolism of the PIs has to be finely regulated both in time and space, and this is achieved by controlling the subcellular distribution, regulation, and activation states of the enzymes involved in their synthesis and removal (kinases and phosphatases). These exist in many different isoforms, each of which appears to have a distinctive intracellular localization and regulation. As a consequence of this subcompartimentalized PI metabolism, a sort of “PI-fingerprint” of each cell membrane compartment is generated. When combined with the targeted recruitment of their protein effectors and the different intracellular distributions of other lipids and regulatory proteins (such as small GTPases), these factors can maintain and determine the identity of the cell organelles despite the extensive membrane flux []. Here, we provide an overview of the regulation and roles of different phosphoinositide kinases and phosphatases and their lipid products at the Golgi complex.
Keywords: Phosphoinositide; Golgi complex; Lipid binding module;

Golgi structure in stress sensing and apoptosis by Stuart W. Hicks; Carolyn E. Machamer (406-414).
The Golgi complex in mammalian cells is composed of polarized stacks of flattened cisternal membranes. Stacks are connected by tubules forming a reticular network of membranes closely associated with the microtubule-organizing center. While the Golgi structure is important for the efficient processing of secretory cargo, the organization of the mammalian Golgi complex may indicate potential functions in addition to the processing and sorting of cargo. Similar to the endoplasmic reticulum stress response pathway, the Golgi complex may initiate signaling pathways to alleviate stress, and if irreparable, trigger apoptosis. Here, we review recent experimental evidence suggesting that the elaborate structure of the Golgi complex in mammalian cells may have evolved to sense and transduce stress signals.
Keywords: Golgi complex; Golgin; Caspase cleavage; Apoptosis; Golgi structure; Signaling;

Clathrin-mediated vesicular trafficking events underpin the vectorial transfer of macromolecules between several eukaryotic membrane-bound compartments. Classical models for coat operation, focused principally on interactions between clathrin, the heterotetrameric adaptor complexes, and cargo molecules, fail to account for the full complexity of the coat assembly and sorting process. New data reveal that targeting of clathrin adaptor complexes is generally supported by phosphoinositides, that cargo recognition by heterotetrameric adaptors depends on phosphorylation-driven conformational alterations, and that dedicated clathrin-associated sorting proteins (CLASPs) exist to promote the selective trafficking of specific categories of cargo. A host of accessory factors also participate in coat polymerization events, and the independently folded appendage domains that project off the heterotetrameric adaptor core function as recruitment platforms that appear to oversee assembly operations. It is also now clear that focal polymerization of branched actin microfilaments contributes to clathrin-coated vesicle assembly and movement at both plasma membrane and Golgi sites. This improved appreciation of the complex mechanisms governing clathrin-dependent sorting events reveals several common principles of clathrin operation at the Golgi and the plasma membrane.
Keywords: Clathrin-coated vesicle; AP-1/AP-2 adaptors; Receptor sorting; Cargo selection; Phosphoinositide; Phosphorylation;

The late Golgi compartment is a major protein sorting station in the cell. Secreted proteins, cell surface proteins, and proteins destined for endosomes or lysosomes must be sorted from one another at this compartment and targeted to their correct destinations. The molecular details of protein trafficking pathways from the late Golgi to the endosomal system are becoming increasingly well understood due in part to information obtained by genetic analysis of yeast. It is now clear that proteins identified in yeast have functional homologues (orthologues) in higher organisms. We will review the molecular mechanisms of protein targeting from the late Golgi to endosomes and to the vacuole (the equivalent of the mammalian lysosome) of the budding yeast Saccharomyces cerevisiae.
Keywords: Vacuole protein sorting (VPS); Multivesicular body (MVB); Endosome; Protein trafficking; Yeast;

Protein sorting in the Golgi complex: Shifting paradigms by Enrique Rodriguez-Boulan; Anne Müsch (455-464).
The paradigms for transport along the biosynthetic route have changed dramatically over the past 15 years. Unlike the situation 15 years ago, the current paradigm involves sorting signals practically at every step of the pathway. In particular, at the exit from the Golgi complex, apical, basolateral and lysosomal targeting signals result in the generation of a variety of routes. Furthermore, it is now quite clear that not all sorting in the biosynthetic route occurs in the Golgi complex or the Trans Golgi Network (TGN). Sorting may occur distally to the Golgi, in recycling endosomes or in budded tubulosaccular structures, or it may occur proximally to the Golgi complex, at the exit from the ER. Several adaptors are candidates to sort apical and basolateral proteins but only AP1B and AP4 are currently involved. Progress is fast and future work should elucidate many of the open questions.
Keywords: Protein trafficking; Polarized secretion; Sorting signal; Golgi complex; Default pathway;

Erratum (465-517).