scholarly journals Endosomal trafficking of yeast membrane proteins

2018 ◽  
Vol 46 (6) ◽  
pp. 1551-1558 ◽  
Author(s):  
Kamilla M. E. Laidlaw ◽  
Chris MacDonald
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Surface Membrane ◽  
Multivesicular Body ◽  
Endosomal Trafficking ◽  
Yeast Saccharomyces Cerevisiae ◽  
Endosomal Membrane ◽  
Recent Developments ◽  
Intracellular Compartments

Various membrane trafficking pathways transport molecules through the endosomal system of eukaryotic cells, where trafficking decisions control the localisation and activity of a diverse repertoire of membrane protein cargoes. The budding yeast Saccharomyces cerevisiae has been used to discover and define many mechanisms that regulate conserved features of endosomal trafficking. Internalised surface membrane proteins first localise to endosomes before sorting to other compartments. Ubiquitination of endosomal membrane proteins is a signal for their degradation. Ubiquitinated cargoes are recognised by the endosomal sorting complex required for transport (ESCRT) apparatus, which mediate sorting through the multivesicular body pathway to the lysosome for degradation. Proteins that are not destined for degradation can be recycled to other intracellular compartments, such as the Golgi and the plasma membrane. In this review, we discuss recent developments elucidating the mechanisms that drive membrane protein degradation and recycling pathways in yeast.

scholarly journals Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Cheng-Wen He ◽  
Xue-Fei Cui ◽  
Shao-Jie Ma ◽  
Qin Xu ◽  
Yan-Peng Ran ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Molecular Mechanisms ◽  
Multivesicular Body ◽  
Degradation Process ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Membrane Structures ◽  
Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

Identification of a Cellubrevin/Vesicle Associated Membrane Protein 3 Homologue in Human Platelets

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Audrey M. Bernstein ◽  
Sidney W. Whiteheart
Keyword(s):  
Membrane Proteins ◽  
Complex Formation ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Human Platelets ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Transport Vesicle ◽  
Exocytic Pathway ◽  
Conserved Core

Abstract Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N–terminal domain. Northern blot analysis showed that the 2.5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in -SNAP–dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most -granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

1997 ◽  
Vol 138 (3) ◽  
pp. 531-545 ◽  
Author(s):  
Robert C. Piper ◽  
Nia J. Bryant ◽  
Tom H. Stevens
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Transmembrane Domain ◽  
Alternative Pathway ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Atpase Subunit ◽  
Transport Vesicles ◽  
Dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

scholarly journals Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

2021 ◽  
Author(s):  
Giedre Ratkeviciute ◽  
Benjamin F. Cooper ◽  
Timothy J. Knowles
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Stability ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Modus Operandi ◽  
Membrane Mimetic ◽  
Aqueous Environments ◽  
Recent Developments

The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.

scholarly journals Recycling of nutrient transporters from yeast endosomes is regulated by ubiquitinated Ist1

2021 ◽  
Author(s):  
Kamilla M Laidlaw ◽  
Grant Calder ◽  
Chris MacDonald
Keyword(s):  
Surface Membrane ◽  
Dynamic Network ◽  
Division Of Labour ◽  
Surface Proteins ◽  
Endosomal Trafficking ◽  
Nutrient Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Recycling Endosomes ◽  
Endosomal Recycling ◽  
Golgi Network

Trafficking of cell surface membrane proteins through a dynamic network of endomembrane compartments ensures correct cellular function. Upon internalisation, many surface proteins are either degraded or recycled back to the plasma membrane. Although these pathways control many biological processes, the precise regulatory features and division of labour between interconnected pathways is unclear. Using the budding yeast Saccharomyces cerevisiae, our work suggests retrograde recycling via the trans-Golgi Network (TGN) of cargoes like yeast synaptobrevins (Snc1 / Snc2), that rely on cargo ubiquitination, is distinct from endosomal trafficking of nutrient transporters. We provide evidence that nutrient transporters internalise to, and upon deubiquitination recycle from, endosomes marked by Vps4 and Ist1. A genetic screen for this recycling pathway previously implicated the ESCRT-III associated factor Ist1, suggesting Ist1 functionally defines yeast recycling endosomes. We demonstrate Ist1 ubiquitination affects its endosomal recruitment and ability to promote recycling. Finally, we reveal the ubiquitin-binding adaptor Npl4 and the essential ATPase Cdc48 are also involved in recycling. Our work suggests features of endosomal recycling are evolutionarily conserved.

Crystallization of Membrane Proteins

1999 ◽  
Author(s):  
F. Reiss-Husson ◽  
D. Picot
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein A ◽  
Limited Proteolysis ◽  
Three Dimensional ◽  
Hydrophobic Surface ◽  
Biological Macromolecules ◽  
Recent Developments ◽  
Tight Association ◽  
Membrane Components

Crystallization of membrane proteins is one of the most recent developments in protein crystal growth; in 1980, for the first time, two membrane proteins were successfully crystallized, bacteriorhodopsin (1) and porin (2). Since then, a number of membrane proteins (about 30) yielded three-dimensional crystals. In several cases, the quality of the crystals was sufficient for X-ray diffraction studies. The first atomic structure of a membrane protein, a photosynthetic bacterial reaction centre, was described in 1985 (3), followed by the structure of about ten other membrane protein families. Crystallization of membrane proteins is now an actively growing field, and has been discussed in several recent reviews (4-8). The major difficulty in the study of membrane proteins, which for years hampered their crystallization, comes from their peculiar solubility properties. These originate from their tight association with other membrane components, particularly lipids. Indeed integral membrane proteins contain hydrophobic surface regions buried in the lipid bilayer core, as well as hydrophilic regions with charged or polar residues more or less exposed at the external faces of the membrane. Disruption of the bilayer for isolating a membrane protein can be done in various ways: extraction with organic solvents, use of chaotropic agents, or solubilization by a detergent. The last method is the most frequently used, since it maintains the biological activity of the protein if a suitable detergent is found. This chapter will be restricted to specific aspects of three-dimensional crystallizations done in micellar solutions of detergent. In some cases, it is possible to separate soluble domains from the membrane protein either by limited proteolysis or by genetic engineering. Such protein fragments can then be treated as soluble proteins and so will not be discussed further in this chapter. We refer to Chapter 12 and the review by Kühlbrandt (9) for the methodology of two-dimensional crystallization used for electron diffraction. The general principles discussed in this book for the crystallization of soluble biological macromolecules apply for membrane proteins; the protein solution must be brought to supersaturation by modifying its physical parameters (concentrations of constituents, ionic strength, and so on), so that nucleation may occur.

scholarly journals Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes

1998 ◽  
Vol 143 (4) ◽  
pp. 957-971 ◽  
Author(s):  
Rytis Prekeris ◽  
Judith Klumperman ◽  
Yu A. Chen ◽  
Richard H. Scheller
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Trafficking ◽  
Transferrin Receptor ◽  
Endosomal Trafficking ◽  
Plasma Membrane Proteins ◽  
Recycling Endosomes ◽  
Confocal Immunofluorescence ◽  
Triton X ◽  
Coated Membrane

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of βSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added αSNAP and NSF and dissociates in the presence of ATP, but not ATPγS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

Identification of a Cellubrevin/Vesicle Associated Membrane Protein 3 Homologue in Human Platelets

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Audrey M. Bernstein ◽  
Sidney W. Whiteheart
Keyword(s):  
Membrane Proteins ◽  
Complex Formation ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Human Platelets ◽  
Degenerate Primers ◽  
Hek 293 ◽  
Transport Vesicle ◽  
Exocytic Pathway ◽  
Conserved Core

Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N–terminal domain. Northern blot analysis showed that the 2.5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in -SNAP–dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most -granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.

scholarly journals Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)

1981 ◽  
Vol 196 (2) ◽  
pp. 531-536 ◽  
Author(s):  
J R Woodward ◽  
H L Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Wild Type ◽  
Amino Acid Permease ◽  
Yeast Saccharomyces Cerevisiae ◽  
General Amino Acid Permease ◽  
Wild Type Yeast

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.

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