Genome-scale modeling of the protein secretory machinery in yeast

The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking. Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands associated with the processing of each specific protein as well as a quantitative estimation of the activity of each component of the secretory machinery

Structural Arrangement and Conformational Dynamics of the γ Subunit of the Na⁺/K⁺-ATPase

The Na+/K+-ATPase couples the chemical energy in ATP to transport Na+ and K+ across the plasma membrane against a concentration gradient. The ion pump is composed of two mandatory subunits: the alpha subunit, which is the major catalytic subunit, and the beta subunit, which is required for proper trafficking of the complex to the plasma membrane. In some tissues, the ion pump also contains an optional third subunit, gamma, which modulates the pump activity. To examine the conformational dynamics of the gamma subunit during ion transport and its position in relation to the alpha and the beta subunits, we have used fluorescence resonance energy transfer under voltage clamp conditions. From these experiments, evidence is provided that the gamma subunit is located adjacent to the M2-M6-M9 pocket of the alpha subunit at the transmembrane-extracellular interface. We have also used fluorescence resonance energy transfer to investigate the relative movement of the three subunits as the ion pump shuttles between the two main conformational states, E1 and E2, as described by the Albers-Post scheme. The results from this study suggest that there is no relative change in distance between the alpha and gamma subunits but there is a relative change in distance between the beta and gamma subunits during the E2 to E1 transition. It was also observed that labeling the gamma subunit at specific residues with fluorophores induces a decrease in K+-induced stationary current. This result could be due to a perturbation in the K+ branch of the reaction cycle of the pump, representing a new way to inhibit the pump

The Accessory Sec Protein Asp2 Modulates GlcNAc Deposition onto the Serine-Rich Repeat Glycoprotein GspB

The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport