Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noneaveolar plasma membrane Similarly, NOS enzymatic activity was 94-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in non-caveolar plasma membrane 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold Thus, acylation targets eNOS to plasmalemmal caveolae Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide

Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae

The αβ-tubulin heterodimer, the building block of microtubules, is subject to a large number of post-translational modifications, comparable in diversity to the intensively studied histone modifications. Although these unusual modifications are conserved throughout evolution, their functions have remained almost completely elusive. Recently, however, important advances in the understanding of how tubulin modifications regulate function and organization have been made.

/pdf/post-translational-modifications-regulate-microtubule-1xsvjaludr.pdf

Post-translational modifications regulate microtubule function

Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.

Microtubules create diverse arrays with specific cellular functions such as the mitotic spindle, cilia and bundles inside neurons. How microtubules are regulated to enable specific functions is not well understood. Recent work has shown that posttranslational modifications of the tubulin building blocks mark subpopulations of microtubules and selectively affect downstream microtubule-based functions. In this way, the tubulin modifications generate a "code" that can be read by microtubule-associated proteins in a manner analogous to how the histone code directs diverse chromatin functions. Here we review recent progress in understanding how the tubulin code is generated, maintained, and read by microtubule effectors.

The tubulin code.

Microtubules are cytoskeletal filaments that are dynamically assembled from α/β-tubulin heterodimers. The primary sequence and structure of the tubulin proteins and, consequently, the properties and architecture of microtubules are highly conserved in eukaryotes. Despite this conservation, tubulin is subject to heterogeneity that is generated in two ways: by the expression of different tubulin isotypes and by posttranslational modifications (PTMs). Identifying the mechanisms that generate and control tubulin heterogeneity and how this heterogeneity affects microtubule function are long-standing goals in the field. Recent work on tubulin PTMs has shed light on how these modifications could contribute to a “tubulin code” that coordinates the complex functions of microtubules in cells.

https://rupress.org/jcb/article-pdf/206/4/461/1365515/jcb_201406055.pdf

The tubulin code: molecular components, readout mechanisms, and functions.

Regulatory nucleotide sequences for a novel Pichia pastoris gene, designated PpSEC10 gene, and the nucleotide sequences and respective amino acid sequences for the secretion leader and the mature Sec10p protein components of the precursor polypeptide encoded by this novel gene are provided. These compositions are useful in methods for expression and secretion of proteins when assembled in proper reading frame, individually or in combination, within a DNA construct that further comprises a nucleotide sequence encoding a protein of interest. Vectors comprising the DNA constructs of the invention can be used to transform a yeast host cell, which can then be cultured to obtain the secreted protein of interest. Kits useful in this method and in methods of detection of the Sec10p protein using antibodies are also disclosed.

Pichia pastoris gene sequences and methods for their use

We generated a strain of Saccharomyces cerevisiae in which the sole source of -tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S -tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15°C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in -tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated -tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated -tubulin in C377S tub1 cells. Our results suggest that cys-377 of -tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.

Single Site α-Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase in Saccharomyces cerevisiae: Possible Role for Palmitoylation of α-Tubulin

Polynucleotides, vectors and host cells comprising a polynucleotide having a fragment of a leader sequence and a second nucleotide sequence that encodes a polypeptide heterologous to the leader sequence, wherein the leader sequence fragment is sufficient for secretion and comprises an amino acid sequence that comprises at least about 70 % sequence identity to the leader sequence of Pichia acaciae killer toxin, wherein the heterologous polypeptide is not naturally contiguous to the leader sequence, and wherein upon expression of the polynucleotide molecule in a host cell suitable for expression thereof, the heterologous polypeptide is produced that is free of additional N-terminal amino acids.

Pichia secretory leader for protein expression

This invention relates to improved methods of recombinant protein production in a variety of host cells including yeast and bacteria. The host cells herein are deficient in protease activity, but the deficiency is not a result of a defective protease structural gene.

Method of making a protease deficient host

Methods and compositions for the expression of nucleotide sequences are provided. Compositions comprise the nucleic acid sequences of the transcriptional regulatory region of the PpSEC10 gene of Pichia pastoris. Methods are provided to chemically regulate the PpSEC20 transcriptiona control region by modulating the iron concentration in he culturing medium. The methods find use in regulating expression of nucleotide sequences. Furthermore, the methods of the invention can be used to regulate polypeptide expression, more particularly in regulating heterologous polypeptide expression, particularly using a yeast host cell as the expression system.

Robert J. Bishop

Papers

Pichia pastoris gene sequences and methods for their use

Single Site α-Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase in Saccharomyces cerevisiae: Possible Role for Palmitoylation of α-Tubulin

Pichia secretory leader for protein expression

Method of making a protease deficient host

Iron regulation of the PpSEC10 transcriptional regulatory region of Pichia pastoris and methods of use