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Olga Martzoukou, Sotiris Amillis, Amalia Zervakou, Savvas Christoforidis, George Diallinas
Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 β subunit (β2) of higher fungi lacks a clathrin-binding domain, and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaB(End4) and SagA(End3) and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae...
February 21, 2017: ELife
Edoardo Petrini, Victoire Baillet, Jake Cridge, Cassandra J Hogan, Cindy Guillaume, Huiling Ke, Elisa Brandetti, Simon Walker, Hashem Koohy, Mikhail Spivakov, Patrick Varga-Weisz
Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1...
October 15, 2015: Journal of Cell Science
Anne M Smardon, Patricia M Kane
Yeast mutants lacking the intracellular V-ATPase proton pump (vma mutants) have reduced levels of the Pma1p proton pump at the plasma membrane and increased levels in organelles including the vacuolar lumen. We examined the mechanism and physiological consequences of Pma1p mislocalization. Pma1p is ubiquitinated in vma mutants, and ubiquitination depends on the ubiquitin ligase Rsp5p and the arrestin-related adaptor protein Rim8p. vma mutant strains containing rsp5 or rim8 mutations maintain Pma1p at the plasma membrane, suggesting that ubiquitination is required for Pma1p internalization...
November 14, 2014: Journal of Biological Chemistry
W Sun, W Liu, Z Zhang, L Xiao, Z Duan, D Liu, Y Xiong, W Zhu, G Lu, X Liu
BACKGROUND AND PURPOSE: Susceptibility-weighted imaging (SWI) is a high spatial resolution technique that can indirectly demonstrate increased cerebral oxygen extraction. Our aim was to assess whether asymmetric cortical vessel sign (ACVS) on SWI could be associated with early neurological deterioration (END) as well as 90-day unfavorable outcome in patients with acute ischaemic stroke. MATERIALS AND METHODS: Consecutive patients with acute middle cerebral artery (MCA) territory infarction were prospectively enrolled...
November 2014: European Journal of Neurology: the Official Journal of the European Federation of Neurological Societies
Yujiro Higuchi, Jun-ya Shoji, Manabu Arioka, Katsuhiko Kitamoto
Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed...
January 2009: Eukaryotic Cell
Francisco J Navarro, Yusé Martín, José M Siverio
Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation...
November 7, 2008: Journal of Biological Chemistry
Stefania Castagnetti, Ralf Behrens, Paul Nurse
The rod-shaped Schizosaccharomyces pombe cell grows in a polarized fashion from opposing ends. Correct positioning of the growth zones is directed by the polarity marker Tea1 located at the cell ends where actin patches accumulate and cell growth takes place. We show that the S. pombe homologue of Saccharomyces cerevisiae SLA2, a protein involved in cortical actin organization and endocytosis, provides a link between the polarity marker and the growth machinery. In wild-type fission yeast cells, this homologue End4/Sla2 is enriched at cell ends during interphase and localizes to a medial ring at cell division, mirroring the actin localization pattern throughout the cell cycle...
May 1, 2005: Journal of Cell Science
Tomoko Iwaki, Naotaka Tanaka, Hiroshi Takagi, Yuko Giga-Hama, Kaoru Takegawa
To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant...
July 30, 2004: Yeast
Pamela K Hanson, Althea M Grant, J Wylie Nichols
At low temperature, the short-chain fluorescent-labeled phospholipids, 1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37 degrees C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole...
July 1, 2002: Journal of Cell Science
Stefanie Krampe, Eckhard Boles
The yeast high-affinity glucose transporters Hxt6p and Hxt7p are rapidly degraded during nitrogen starvation in the presence of high concentrations of fermentable carbon sources. Our results suggest that degradation is mainly due to the stimulation of general protein turnover and not caused by a mechanism specifically triggered by glucose. Analysis of Hxt6p/7p stability and cellular distribution in end4, aut2 and apg1 mutants indicates that Hxt7p is internalized by endocytosis, and autophagy is involved in the final delivery of Hxt7p to the vacuole for proteolytic degradation...
February 27, 2002: FEBS Letters
E E Luk, V C Culotta
Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished in smf2Delta mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria...
December 14, 2001: Journal of Biological Chemistry
X Gong, A Chang
Pma1 is a plasma membrane H(+)-ATPase whose activity at the cell surface is essential for cell viability. In this paper we describe a temperature-sensitive pma1 allele, pma1-10 (with two point mutations in the first cytoplasmic loop of Pma1), in which the newly synthesized mutant protein fails to remain stable at the cell surface at 37 degrees C. Instead, Pma1-10 appears to undergo internalization for vacuolar degradation in a manner dependent on End4, Vps27, Doa4, and Pep4. By contrast with wild-type Pma1, mutant Pma1-10 is hypophosphorylated and fails to associate with a Triton-insoluble fraction at 37 degrees C, suggesting failure to enter lipid rafts...
July 31, 2001: Proceedings of the National Academy of Sciences of the United States of America
L Li, O S Chen, D McVey Ward, J Kaplan
The budding yeast Saccharomyces cerevisiae can grow for generations in the absence of exogenous iron, indicating a capacity to store intracellular iron. As cells can accumulate iron by endocytosis we studied iron metabolism in yeast that were defective in endocytosis. We demonstrated that endocytosis-defective yeast (Delta end4) can store iron in the vacuole, indicating a transfer of iron from the cytosol to the vacuole. Using several different criteria we demonstrated that CCC1 encodes a transporter that effects the accumulation of iron and Mn(2+) in vacuoles...
August 3, 2001: Journal of Biological Chemistry
G Wang, J M McCaffery, B Wendland, S Dupré, R Haguenauer-Tsapis, J M Huibregtse
The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated...
May 2001: Molecular and Cellular Biology
J Palecek, J Hasek, H Ruis
The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3). Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated. They overlapped in the region of amino acids 318-550. Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction. The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction...
April 20, 2001: Biochemical and Biophysical Research Communications
A M Grant, P K Hanson, L Malone, J W Nichols
The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked...
January 2001: Traffic
T Beck, A Schmidt, M N Hall
In Saccharomyces cerevisiae, amino acid permeases are divided into two classes. One class, represented by the general amino acid permease GAP1, contains permeases regulated in response to the nitrogen source. The other class, including the high affinity tryptophan permease, TAT2, consists of the so-called constitutive permeases. We show that TAT2 is regulated at the level of protein stability. In exponentially growing cells, TAT2 is in the plasma membrane and also accumulates in internal compartments of the secretory pathway...
September 20, 1999: Journal of Cell Biology
U Marx, T Polakowski, T Pomorski, C Lang, H Nelson, N Nelson, A Herrmann
Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids...
July 1999: European Journal of Biochemistry
S Krampe, O Stamm, C P Hollenberg, E Boles
After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis...
December 28, 1998: FEBS Letters
M Ziman, J S Chuang, M Tsung, S Hamamoto, R Schekman
Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Delta strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells...
June 1998: Molecular Biology of the Cell
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