Iron-sulfur-protein biogenesis in eukaryotes

Iron-sulfur (Fe-S) clusters (ISCs) are versatile, ancient co-factors of proteins that are involved in electron transport, enzyme catalysis and regulation of gene expression. The synthesis of ISCs and their insertion into apoproteins involves the function of complex cellular machineries. In eukaryotes, the mitochondrial ISC-assembly machinery is involved in the maturation of all cellular iron-sulfur proteins. A mitochondrial export machinery and a recently discovered cytosolic assembly system specifically participate in the maturation of cytosolic and nuclear iron-sulfur proteins. Of the approximately 20 assembly components, more than ten are encoded by essential genes, which indicates that the process is indispensable for life. Mutations in two of the assembly components lead to neurological diseases. The essential character of Fe-S-protein biogenesis in eukaryotes and its importance for human disease identifies this evolutionary ancient process as one of the most important biosynthetic pathways of life.     

Golgi biogenesis

The Golgi is an essential membrane-bound organelle in the secretary pathway of eukaryotic cells. In mammalian cells, the Golgi stacks are integrated into a continuous perinuclear ribbon, which poses a challenge for the daughter cells to inherit this membrane organelle during cell division. To facilitate proper partitioning, the mammalian Golgi ribbon is disassembled into vesicles in early mitosis. Following segregation into the daughter cells, a functional Golgi is reformed. Here we summarize our current understanding of the molecular mechanisms that control the mitotic Golgi disassembly and postmitotic reassembly cycle in mammalian cells.     

Ribosomal RNA processing and ribosome biogenesis in eukaryotes

In eukaryotes nearly 500 rRNAs, ribosomal proteins, snoRNAs and trans-acting factors contribute to ribosome biogenesis. After more than 30 years of intense research, the incredible complexities of nucleolar function are revealed but details often remain unclear. Here we review this progress and the many intriguing questions which remain.     

Cryo-EM insights into tail-anchored membrane protein biogenesis in eukaryotes

Tail-anchored (TA) proteins are a biologically significant class of membrane proteins, which require specialised cellular pathways to insert their single C-terminal transmembrane domain into the correct membrane. Cryo-electron microscopy has recently provided new insights into the organelle-specific machineries for TA protein biogenesis. Structures of targeting and insertase complexes within the canonical guided entry of TA proteins (GET) pathway indicate how substrates are faithfully chaperoned into the endoplasmic reticulum (ER) membrane in metazoans. The core of the GET insertase is conserved within structures of the ER membrane protein complex (EMC), which acts in parallel to insert a different subset of TA proteins. Furthermore, structures of the dislocases Spf1 and Msp1 show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes respectively.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Molecular basis for Golgi maintenance and biogenesis

The Golgi apparatus contains thousands of different types of integral and peripheral membrane proteins, perhaps more than any other intracellular organelle. To understand these proteins' roles in Golgi function and in broader cellular processes, it is useful to categorize them according to their contribution to Golgi creation and maintenance. This is because all of the Golgi's functions derive from its ability to maintain steady-state pools of particular proteins and lipids, which in turn relies on the Golgi's dynamic character - that is, its ongoing state of transformation and outgrowth from the endoplasmic reticulum. Here, we categorize the expanding list of Golgi-associated proteins on the basis of their role in Golgi reformation after the Golgi has been disassembled. Information gained on how different proteins participate in this process can provide important insights for understanding the Golgi's global functions within cells.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

Low-molecular-weight RNA of eukaryotes: biogenesis, subcellular localization, functions

The experimental data on the organization and expression of the low-molecular-weight RNA genes on the subcellular localization, metabolism and evolution of these RNA are summarized. The processes in which they are involved are discussed. The biological role of the low-molecular-weight RNA is paid a special attention. A hypothesis concerning the involvement of these molecules in the mechanism of active transport of proteins from the cytoplasm into the nucleus of the cell is put forward.     

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.     

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D ‐ceramide‐C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D ‐ceramide‐C6‐treated HeLa cells revealed an increase in the levels of C6‐sphingomyelin, C6‐glucosylceramide, and diacylglycerol. D ‐ceramide‐C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D ‐ceramide‐C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6‐sphingomyelin prevented liquid‐ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.     

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

p97/p47-Mediated biogenesis of Golgi and ER

In mammalian cells, the Golgi apparatus and endoplasmic reticulum have typical structures during interphase: stacked cisternae located adjacent to the nucleus and a network of interconnected tubules throughout the cytoplasm, respectively. At mitosis their architectures disappear and are reassembled in daughter cells. p97, an AAA-ATPase, mediates membrane fusion and is required for reassembly of these organelles. In the p97-mediated membrane fusion, p47 was identified as an essential cofactor, through which p97 binds to a SNARE, syntaxin5. A second essential cofactor, VCIP135, was identified as a p97/p47/syntaxin5-interacting protein. Several lines of recent evidence suggest that ubiquitination may be implicated in the p97/p47 pathway; p47 binds to monoubiquitinated proteins and VCIP135 shows a deubiquitinating activity in vitro. For the cell-cycle regulation of the p97/p47 pathway, it has been reported that the localization and phosphorylation-dephosphorylation of p47 are crucial. In this review, we describe the components involved in the p97-mediated membrane fusion and discuss the regulation of the fusion pathway.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.     

Cyanidioschyzon merolae genome. A tool for facilitating comparable studies on organelle biogenesis in photosynthetic eukaryotes

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.     

Building a secretory apparatus: role of ARF1/COPI in Golgi biogenesis and maintenance

 The secretory apparatus within all eukaryotic cells comprises a dynamic membrane system with bidirectional membrane transport pathways and overlapping compartmental boundaries. Membrane traffic and organelle biogenesis/maintenance are fundamentally linked within this system, with perturbations in membrane traffic quickly leading to changes in organelle structure and identity. Dissection of the molecular basis of these properties in yeast and mammalian cells has revealed a crucial role for the cytoplasmic protein complex ARF1/COPI, which undergoes regulated assembly and disassembly with membranes. ARF1/COPI appears to be involved in the formation and maintenance of the Golgi complex, which is the receiving and delivery station for all secretory traffic. ARF1-GTP, through assembly of COPI to membranes and, possibly, through activation of PLD, is likely to promote the formation and maturation of pre-Golgi intermediates into Golgi elements, whereas ARF-GDP causes COPI dissociation and stimulates the formation of retrograde transport structures that recycle Golgi membrane back to the ER. These processes are appear to underlie the coupling of organelle biogenesis and membrane trafficking within cells, allowing the size and shape of secretory organelles to be altered in response to changing cellular needs. Future work needs to address how the activation and localization of ARF1/COPI to membranes as well as other related factors are temporally and spatially regulated, and by what mechanism they transform membrane shape and dynamics to facilitate protein transport and compartmental functioning. Accepted: 23 March 1998