A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.

The association of glycolytic enzymes from yeast confers resistance against inhibition by trehalose.

During stress, many organisms accumulate compatible solutes. These solutes must be eliminated upon return to optimal conditions as they inhibit cell metabolism and growth. In contrast, enzyme interactions optimize metabolism through mechanisms such as channeling of substrates. It was decided to test the (compatible solute) trehalose-mediated inhibition of some yeast glycolytic pathway enzymes known to associate and whether inhibition is prevented when enzymes are allowed to associate. Trehalose inhibited the isolated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hexokinase (HXK), but not aldolase (ALD) nor phosphoglycerate kinase (PGK). When these enzymes were mixed in pairs, both GAPDH and HXK were protected by either ALD or PGK acquiring the inhibition behavior of the resistant enzyme. GAPDH was not protected by HXK, albumin or lactate dehydrogenase (LDH). Also, ALD did not protect glucose 6-phosphate dehydrogenase (G6PDH), suggesting that protection is specific. In yeast cell extracts, fermentation was resistant to trehalose inhibition, suggesting all enzymes involved in the glucose-dependent production of ethanol were stabilized. It is suggested that during the yeast stress response, enzyme association protects some metabolic pathways against trehalose-mediated inhibition.

In guinea pig sperm, aldolase A forms a complex with actin, WAS, and Arp2/3 that plays a role in actin polymerization.

Glycolytic enzymes have, in addition to their role in energy production, other functions in the regulation of cellular processes. Aldolase A has been reported to be present in sperm, playing a key role in glycolysis; however, despite its reported interactions with actin and WAS, little is known about a non-glycolytic role of aldolase A in sperm. Here, we show that in guinea pig spermatozoa, aldolase A is tightly associated to cytoskeletal structures where it interacts with actin, WAS, and Arp2/3. We show that aldolase A spermatozoa treatment increases their polymerized actin levels. In addition, we show that there is a direct correlation between the levels of polymerized actin and the levels of aldolase A-actin interaction. Our results suggest that aldolase A functions as a bridge between filaments of actin and the actin-polymerizing machinery.

In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction.

A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio-temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.

Perinuclear theca during spermatozoa maturation leading to fertilization.

Mammalian spermatozoa acquire the capacity to fertilize the ovum and display motility during their passage through the epididymis. At the same time, they undergo changes in metabolic patterns, enzymatic activities, ability to bind to zona pellucida surface, and electrophoretic properties and, furthermore, stabilization of some sperm structures by the establishment of disulphide linkages takes place in several sperm structures. The cytoplasmic perinuclear theca (PT) is a unique extranuclear cytoskeletal element that surrounds the nucleus, which is proposed to be a structural scaffold to the sperm nucleus. The purpose of this review is to describe PT changes related to epididymal sperm maturation. We will focus mainly on the protein components of the PT of eutherian mammalian spermatozoa and on quantitative protein changes during sperm maturation. The protein constituents of the PT have not been completely defined and most of them are different from the cytoskeletal proteins of somatic cells. However, they are proteins with cytoskeletal features. The morphologic changes reported for PT and the proposed functions of PT are discussed.