Atg9 proteins,not so different after all

Macroautophagy (hereafter autophagy) is a catabolic pathway present in all eukaryotic cells. The yeast Saccharomyces cerevisiae has been pivotal in the identification and characterization of the key autophagy-related (Atg) proteins, which play a central role in the generation of autophagosomes. The components of the core Atg/ATG machinery and their functions are highly conserved among species, although mammalian cells also have isoforms and auxiliary factors. Atg9/ATG9 is the only transmembrane protein that is part of the core Atg/ATG machinery, but it appears to have divergent localizations and molecular roles in yeast and mammals. A recent experimental analysis of the yeast endo-lysosomal system by the laboratory of Benjamin Glick, however, suggests a more simple organization of this membrane system. Although this study has not examined yeast Atg9, its findings place this protein in the same compartments as its mammalian counterpart. Here, we will discuss the implications of this conceptual change on the trafficking of yeast Atg9 and its function in autophagy.     

Differential requirements of mammalian ESCRTs in multivesicular body formation, virus budding and cell division

The endosomal sorting complexes required for transport (ESCRTs) mediate membrane fission from the cytoplasmic face of the bud neck. ESCRTs were originally identified as factors involved in multivesicular body vesicle biogenesis in yeast but have since been shown to function in other membrane fission events in mammalian cells, including enveloped virus budding and the abscission step of cytokinesis. Several recent studies have revealed that not all ESCRT factors are required for each of these biological processes, and this review summarizes our current understanding of the different requirements for ESCRT factors in these three different ESCRT-mediated mammalian membrane fission processes.     

Calcium homeostasis and signaling in yeast cells and cardiac myocytes

Calcium ions are the most ubiquitous and versatile signaling molecules in eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both the normal growth of the budding yeast Saccharomyces cerevisiae and the intricate working of the mammalian heart. In this paper, we make a detailed comparison between the calcium homeostasis/signaling networks in yeast cells and those in mammalian cardiac myocytes. This comparison covers not only the components, structure and function of the networks but also includes existing knowledge on the measured and simulated network dynamics using mathematical models. Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate similarly in mammalian cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is a simple organism that affords powerful genetic and genomic tools. Thus, exploring and understanding the calcium homeostasis/signaling system in yeast can provide a shortcut to help understand calcium homeostasis/signaling systems in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.     

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.     

Mitochondrial death pathways in yeast and mammalian cells

In mammals, mitochondria are important mediators of programmed cell death, and this process is often regulated by Bcl-2 family proteins. However, a role for mitochondria-mediated cell death in non-mammalian species is more controversial. New evidence from a variety of sources suggests that mammalian mitochondrial fission/division proteins also have the capacity to promote programmed cell death, which may involve interactions with Bcl-2 family proteins. Homologues of these fission factors and several additional mammalian cell death regulators are conserved in flies, worms and yeast, and have been suggested to regulate programmed cell death in these species as well. However, the molecular mechanisms by which these phylogenetically conserved proteins contribute to cell death are not known for any species. Some have taken the conserved pro-death activity of mitochondrial fission factors to mean that mitochondrial fission per se, or failed attempts to undergo fission, are directly involved in cell death. Other evidence suggests that the fission function and the cell death function of these factors are separable. Here we consider the evidence for these arguments and their implications regarding the origins of programmed cell death.     

Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3- D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP- N- acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N- acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N- glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c- Myc epitope tag at the C-terminus elucidates that the N - terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.      

MicroReview Control of translation initiation in Saccharomyces cerevisiae

The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of 'Sherman's Rules' for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5' end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1(-) yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1(-) yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.     

Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7

Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.