scholarly journals Saccharomyces cerevisiae Aqr1 Is an Internal-Membrane Transporter Involved in Excretion of Amino Acids

2004 ◽  
Vol 3 (6) ◽  
pp. 1492-1503 ◽  
Author(s):  
Isabel Velasco ◽  
Sandra Tenreiro ◽  
Isabel L. Calderon ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Negative Control ◽  
Yeast Cells ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Membrane Structures ◽  
Internal Membrane ◽  
Green Fluorescent

ABSTRACT Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms. It is typically favored by overproduction of the amino acid and/or impairment of its uptake. Here we describe the construction of a yeast strain excreting threonine and homoserine. Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H+ antiport, is involved in homoserine and threonine excretion. Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol. Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4. An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane. The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells. We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis.

scholarly journals Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

2001 ◽  
Vol 75 (19) ◽  
pp. 9393-9406 ◽  
Author(s):  
Michael M. Goodin ◽  
Jennifer Austin ◽  
Renée Tobias ◽  
Miki Fujita ◽  
Christina Morales ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Protein Fusion ◽  
N Protein ◽  
Subnuclear Localization ◽  
P Proteins ◽  
Green Fluorescent

ABSTRACT We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathioneS-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.

scholarly journals Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

2005 ◽  
Vol 187 (10) ◽  
pp. 3572-3580 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Bertolt Gust ◽  
Jolanta Zakrzewska-Czerwinska ◽  
Keith F. Chater
Keyword(s):  
Green Fluorescent Protein ◽  
Developmental Stage ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Chromosome Partitioning ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

Induction of the arginine vasopressin-enhanced green fluorescent protein fusion transgene in the rat locus coeruleus

Stress ◽  
2010 ◽  
Vol 13 (4) ◽  
pp. 281-292 ◽  
Author(s):  
Miwako Todoroki ◽  
Yoichi Ueta ◽  
Hiroaki Fujihara ◽  
Hiroki Otsubo ◽  
Minori Shibata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Locus Coeruleus ◽  
Arginine Vasopressin ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent

scholarly journals Yeast Exocytic v-SNAREs Confer Endocytosis

2000 ◽  
Vol 11 (10) ◽  
pp. 3629-3643 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Daphne Chapman-Shimshoni ◽  
Selena Trajkovic ◽  
Jeffrey E. Gerst
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Metabolic Labeling ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Direct Role ◽  
Endosomal Transport ◽  
Sensitive Allele ◽  
Green Fluorescent

In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1(SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that bothSNC and temperature-shiftedSNC1ala43yeast are deficient in their ability to deliver the soluble dye FM4–64 to the vacuole. Under conditions in which vesicles accumulate, FM4–64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, α-factor–stimulated endocytosis of the α-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1ala43was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.

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