Deletion of PIN4 Suppresses the Protein Transport Defects Caused by sec12-4 Mutation in Saccharomyces cerevisiae

2020 ◽  
Vol 30 (1-6) ◽  
pp. 25-35
Author(s):  
Akiko Murakami-Sekimata ◽  
Masayuki Sekimata ◽  
Natsumi Sato ◽  
Yuto Hayasaka ◽  
Akihiko Nakano
Keyword(s):  
Protein Transport ◽  
Cell Cycle Checkpoint ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Casein Kinase I ◽  
Vesicle Budding ◽  
Nucleotide Exchange Factor ◽  
Cycle Checkpoint ◽  
Copii Vesicles

Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of <i>sec12-4</i>, <i>PIN4/MDT1</i>, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of <i>PIN4</i> suppressed both defects of temperature-sensitive growth and the partial protein transport observed in <i>sec12-4</i> mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.

scholarly journals The Ran GTPase System in Fission Yeast Affects Microtubules and Cytokinesis in Cells That Are Competent for Nucleocytoplasmic Protein Transport

2002 ◽  
Vol 22 (24) ◽  
pp. 8491-8505 ◽  
Author(s):  
Sandra S. Salus ◽  
Janos Demeter ◽  
Shelley Sazer
Keyword(s):  
Fission Yeast ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Nucleotide Exchange Factor ◽  
Ring Structures ◽  
Dependent Processes ◽  
In Cells

ABSTRACT Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

scholarly journals Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

2014 ◽  
Vol 206 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Kota Saito ◽  
Koh Yamashiro ◽  
Noriko Shimazu ◽  
Tomoya Tanabe ◽  
Kenji Kontani ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Secretory Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Transport Vesicles ◽  
Nucleotide Exchange Factor ◽  
Receptor Component ◽  
Er Exit Sites ◽  
Guanosine Triphosphatase ◽  
Trimeric Structure

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae

1996 ◽  
Vol 109 (9) ◽  
pp. 2311-2318 ◽  
Author(s):  
N. Nakashima ◽  
N. Hayashi ◽  
E. Noguchi ◽  
T. Nishimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Importin Alpha ◽  
Nucleotide Exchange Factor ◽  
Cold Sensitive ◽  
Gtpase Cycle

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

scholarly journals The Yeast ULP2 (SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein

2000 ◽  
Vol 20 (7) ◽  
pp. 2367-2377 ◽  
Author(s):  
Shyr-Jiann Li ◽  
Mark Hochstrasser
Keyword(s):  
Cell Function ◽  
Feedback Mechanism ◽  
Cell Cycle Checkpoint ◽  
Temperature Sensitive ◽  
Partial Recovery ◽  
Protein Ligation ◽  
Protein Conjugates ◽  
Dna Damaging Agents ◽  
Cycle Checkpoint ◽  
Specific Protease

ABSTRACT Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. LikeSMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1ts strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Δ mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of aulp1ts mutation or overproduction of catalytically inactive Ulp1 can substantially overcome theulp2Δ defects. Inactivation of Ulp2 also suppresses several ulp1ts defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

scholarly journals Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1132
Author(s):  
Xiaolong Wang ◽  
Meiqian Weng ◽  
Yuting Ke ◽  
Ellen Sapp ◽  
Marian DiFiglia ◽  
...  
Keyword(s):  
Low Frequency ◽  
Cell Movement ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Exchange ◽  
Vesicle Budding ◽  
Recycling Endosomes ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Nucleotide Exchange Factor ◽  
Elevated Expression

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

scholarly journals Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

2015 ◽  
Vol 112 (25) ◽  
pp. E3199-E3206 ◽  
Author(s):  
Kanika Bajaj Pahuja ◽  
Jinzhi Wang ◽  
Anastasia Blagoveshchenskaya ◽  
Lillian Lim ◽  
M. S. Madhusudhan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Direct Interaction ◽  
Phosphatidyl Inositol ◽  
Adaptor Proteins ◽  
Secretory Proteins ◽  
Vesicle Budding ◽  
Cargo Proteins ◽  
A Cell ◽  
Copii Vesicles ◽  
Serum Dependent

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.

scholarly journals cTAGE5 acts as a Sar1 GTPase regulator for collagen export

2018 ◽  
Author(s):  
Norito Sasaki ◽  
Masano Shiraiwa ◽  
Miharu Maeda ◽  
Tomohiro Yorimitsu ◽  
Ken Sato ◽  
...  
Keyword(s):  
Small Gtpase ◽  
Coated Vesicles ◽  
Secretory Proteins ◽  
Efficient Production ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Collagen Secretion ◽  
Nucleotide Exchange Factor ◽  
Er Exit Sites

AbstractSecretory proteins synthesized within the endoplasmic reticulum (ER) are exported via coat protein complex II (COPII)-coated vesicles. The formation of the COPII-coated vesicles is initiated by activation of the small GTPase, Sar1. cTAGE5 directly interacts with a guanine-nucleotide exchange factor (GEF), Sec12, and a GTPase-activating protein (GAP) of Sar1, Sec23. We have previously shown that cTAGE5 recruits Sec12 to the ER exit sites for efficient production of activated Sar1 for collagen secretion. However, the functional significance of the interaction between cTAGE5 and Sec23 has not been fully elucidated. In this study, we showed that cTAGE5 enhances the GAP activity of Sec23 toward Sar1. In addition, the interaction of cTAGE5 with Sec23 is necessary for collagen exit from the ER. Our data suggests that cTAGE5 acts as a Sar1 GTPase regulator for collagen secretion.

Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation

1998 ◽  
Vol 111 (7) ◽  
pp. 977-984
Author(s):  
A.A. Sablina ◽  
G.V. Ilyinskaya ◽  
S.N. Rubtsova ◽  
L.S. Agapova ◽  
P.M. Chumakov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Cell Activation ◽  
P53 Gene ◽  
Cell Cycle Checkpoint ◽  
Abnormal Chromosome ◽  
Brdu Incorporation ◽  
Temperature Sensitive ◽  
Cells With Micronuclei ◽  
Cycle Checkpoint

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.

scholarly journals The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

2001 ◽  
Vol 12 (4) ◽  
pp. 1035-1045 ◽  
Author(s):  
Anne Spang ◽  
Johannes M. Herrmann ◽  
Susan Hamamoto ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Guanine Nucleotide Exchange Factors ◽  
Temperature Sensitive ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Membrane Bound

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identifiedGEA2 as a multicopy suppressor of asec21-3 temperature-sensitive mutant.SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat.GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 andGEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

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