scholarly journals Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast

2006 ◽  
Vol 175 (6) ◽  
pp. 925-935 ◽  
Author(s):  
Congcong He ◽  
Hui Song ◽  
Tomohiro Yorimitsu ◽  
Iryna Monastyrska ◽  
Wei-Lien Yen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Actin Cytoskeleton ◽  
Integral Membrane Protein ◽  
Yeast Two Hybrid ◽  
Anterograde Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
Selective Autophagy ◽  
Peripheral Membrane Protein ◽  
Developmental Conditions ◽  
Two Hybrid

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid–based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth.

scholarly journals The peroxin Pex34p functions with the Pex11 family of peroxisomal divisional proteins to regulate the peroxisome population in yeast

2011 ◽  
Vol 22 (10) ◽  
pp. 1727-1738 ◽  
Author(s):  
Robert J. Tower ◽  
Andrei Fagarasanu ◽  
John D. Aitchison ◽  
Richard A. Rachubinski
Keyword(s):  
Integral Membrane Protein ◽  
Protein Family ◽  
Matrix Proteins ◽  
Peroxisome Biogenesis ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Factors ◽  
Peroxisome Division ◽  
Two Hybrid

Peroxisomes are ubiquitous organelles involved in diverse metabolic processes, most notably the metabolism of lipids and the detoxification of reactive oxygen species. Peroxisomes are highly dynamic and change in size and number in response to both intra- and extracellular cues. In the yeast Saccharomyces cerevisiae, peroxisome growth and division are controlled by both the differential import of soluble matrix proteins and a specialized divisional machinery that includes peroxisome-specific factors, such as members of the Pex11 protein family, and general organelle divisional factors, such as the dynamin-related protein Vps1p. Global yeast two-hybrid analyses have demonstrated interactions between the product of the S. cerevisiae gene of unknown function, YCL056c, and Pex proteins involved in peroxisome biogenesis. Here we show that the protein encoded by YCL056c, renamed Pex34p, is a peroxisomal integral membrane protein that acts independently and also in concert with the Pex11 protein family members Pex11p, Pex25p, and Pex27p to control the peroxisome populations of cells under conditions of both peroxisome proliferation and constitutive peroxisome division. Yeast two-hybrid analysis showed that Pex34p interacts physically with itself and with Pex11p, Pex25p, and Pex27p but not with Vps1p. Pex34p can act as a positive effector of peroxisome division as its overexpression leads to increased numbers of peroxisomes in wild type and pex34Δ cells. Pex34p requires the Pex11 family proteins to promote peroxisome division. Our discovery of Pex34p as a protein involved in the already complex control of peroxisome populations emphasizes the necessity of cells to strictly regulate their peroxisome populations to be able to respond appropriately to changing environmental conditions.

scholarly journals Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

1996 ◽  
Vol 16 (2) ◽  
pp. 593-602 ◽  
Author(s):  
R Candau ◽  
P A Moore ◽  
L Wang ◽  
N Barlev ◽  
C Y Ying ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Sequence Similarity ◽  
Adaptor Proteins ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Evolutionarily Conserved ◽  
Human Proteins ◽  
Two Hybrid

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

scholarly journals Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

2008 ◽  
Vol 191 (1) ◽  
pp. 333-346 ◽  
Author(s):  
Gouzel Karimova ◽  
Carine Robichon ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Dependent Manner ◽  
E Coli ◽  
Division Site ◽  
Inner Membrane Protein ◽  
Two Hybrid

ABSTRACT Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

Lipoxygenase Forms Located at the Plant Plasma Membrane

1995 ◽  
Vol 50 (1-2) ◽  
pp. 29-36 ◽  
Author(s):  
Arndt Nellen ◽  
Barbara Rojahn ◽  
Helmut Kindl
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lipid Body ◽  
Integral Membrane Protein ◽  
Long Distance ◽  
Two Phase ◽  
Cucumis Sativus L ◽  
Sds Page ◽  
Peripheral Membrane Protein ◽  
Two Phase Partition

Abstract In cucumber cotyledons (Cucumis sativus L.) containing several soluble and particulate forms of lipoxygenase (LOX), the location of LOX forms in microsomes has been studied. We concentrated on the question whether the plasma membrane contains one or more forms of LOX. As methodology, we applied both the two-phase partition with polyethylene glycol/dextran and density gradient flotation of plasma membrane-enriched membrane fractions. Both methods show that a high percentage of the microsomal LOX can be attributed to the plasma membrane. Emphasis was put on the findings that the LOX located at the plasma membrane consisted of a species behaving as an integral membrane protein and another form characterized as a peripheral membrane protein by solubilization with carbonate. With long distance SDS-PAGE and immunodecoration using anti-lipid body LOX antiserum, it is possible to distinguish between microsomal LOX forms by small but significant differences in size. Treatment of seedlings with methyl jasmonate led to an enhanced level of LOX at the plasma membrane.

scholarly journals Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3zeta

2003 ◽  
Vol 369 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Jörg BIRKENFELD ◽  
Heinrich BETZ ◽  
Dagmar ROTH
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Actin Binding ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Glutathione S Transferase ◽  
Physiological Processes ◽  
Interaction Partners ◽  
Induced Changes ◽  
Two Hybrid

Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3ζ isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3ζ. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3ζ inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3ζ-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1.

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein

1990 ◽  
Vol 10 (8) ◽  
pp. 3873-3883
Author(s):  
M R Mitts ◽  
D B Grant ◽  
W Heideman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Adenylate Cyclase ◽  
Adenylate Cyclase System ◽  
Hydrodynamic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Peripheral Membrane Protein ◽  
Mammalian Enzyme ◽  
Regulatory Significance ◽  
Stimulation Of

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.

scholarly journals Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit

2001 ◽  
Vol 12 (12) ◽  
pp. 3783-3796 ◽  
Author(s):  
Barbara A. Reilly ◽  
Bryan A. Kraynack ◽  
Susan M. VanRheenen ◽  
M. Gerard Waters
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Genetic Interaction ◽  
Target Site ◽  
Restrictive Temperature ◽  
Multiprotein Complex ◽  
Anterograde Transport ◽  
Interacting Protein ◽  
Peripheral Membrane Protein ◽  
Retrograde Traffic

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show thatdsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of ∼80 and ∼55 kDa. The ∼80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is ∼50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the δ-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.

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