Variation in blood serum antifreeze activity of Antarctic Trematomus fishes across habitat temperature and depth.

High latitude waters in the Southern Ocean can be near their freezing point and remain ice-covered throughout the year whereas lower latitude Southern Ocean waters have seasonal ice coverage and comparatively large (6 °C) annual temperature changes. The genus Trematomus (suborder Notothenioidei) is regarded primarily as a high latitude group because of its abundance there, they also inhabit the warmer regions in smaller numbers. Freeze avoidance in the notothenioids is linked to the presence of two antifreeze proteins (AFPs); the antifreeze glycoproteins (AFGPs) and antifreeze potentiating protein (AFPP), both of which adsorb to internal ice crystals inhibiting growth. Both high and low latitude trematomids possess sufficient AFP to lower their blood freezing point below that of seawater (-1.9 °C). We investigated the contributions of AFGPs and AFPP to the blood freezing point depression to determine how they varied with depth, water temperature, and the presence of ice. High latitude trematomids had lower blood freezing points than those inhabiting lower latitude waters indicating differences in their freeze avoidance capacities. Lower freezing points were associated with higher levels of antifreeze activity due to higher levels of both AFGP and AFPP. Populations of Trematomus hansoni and Trematomus bernacchii from shallow depths appear more freeze avoidant than populations inhabiting deep, ice-free water based on their lower freezing points and higher antifreeze activities. Gel electrophoresis of the trichloroacetic acid-soluble AFGPs indicates that only high molecular weight isoforms, which contribute more to AFGP activity, vary across species as well as between individuals of a species.

Constitutive secretion in Tetrahymena thermophila.

The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates.

The effect of carbon source on the secretome of Kluyveromyces lactis.

A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by Kluyveromyces lactis cells. Here, we report changes detected in the K. lactis secretome as a result of growth in three different carbon sources: glucose, galactose and glycerol. A total of 151 secreted proteins were detected by multi-dimensional separations and reversed-phase online nanoESI-MS/MS analysis. From these, we were able to identify 63 proteins (termed the "base secretome") that were common to all three fermentation conditions. The majority of base secretome proteins, 79%, possessed general secretory pathway (GSP) sequences and were involved with cell wall structure, glycosylation, carbohydrate metabolism and proteolysis. There was little variation in the functional groupings of base secretome GSP proteins and GSP proteins that were not part of the base secretome. In contrast, the majority of non-GSP proteins detected were not part of the base secretome and the functions of these proteins varied significantly. Finally, through further identification of non-GSP proteins in carbon sources not originally tested, we have gained further evidence of a protein export mechanism separate from the GSP in K. lactis.