Cyanobacterial leader peptides for protein secretion

The leader peptide of the major secreted protein PilA1 of the cyanobacterium Synechocystis sp. strain PCC 6803 and several artificial leader peptides have been used to study secretion of the reporter protein lichenase to the culture medium. The strains of Synechocystis carrying lichenase with the leader sequences of PilA and with the leader sequence of Slr2016 efficiently secreted the reporter protein. The artificial leader sequence that was characterized by the overall positive charge (as PilA1 and Slr2016 leaders) also allowed secretion. The artificial leader with negative charge, however, did not allow secretion of the reporter protein. Moreover, no secreted proteins have been isolated from this strain using conventional techniques for preparation of secreted proteins. These data suggest that the general secretion pathway in cyanobacteria, at least for pilins, recognizes the overall charge of the leader sequences, and operates in a sequence-non-specific manner.     

The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20°C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44°C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.     

Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803

We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.     

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.     

Changes in Lipid Metabolism during Adaptation of the Dunaliella salina Photosynthetic Apparatus to High CO2 Concentration

The effects of CO₂ on the content and composition of lipid fatty acids (FA) and on the photosynthetic characteristics of unicellular halophilic green alga Dunaliella salina (known to be susceptible to CO₂ stress) were investigated. It was shown that even one-day-long increase in the CO₂ concentration (from 2 to 10%) provoked an increase in the total amount of FA on the dry weight basis by 30%. After 7-day-long growth at 10% CO₂, this value was 2.7-fold higher than that at 2% CO₂. The difference in the FA content and composition indicated the activation of FA synthesis de novo and inhibition of their elongation and desaturation, as well as the increase in the relative content of saturated FA at 10% CO₂. It was demonstrated that, after one-day-long CO₂ stress, the MGDG/DGDG ratio increased fourfold without change in the sum of their FA, which indicates the increase in the proportion of lipids predisposed to micellar (hexagonal phase) but not lamellar structure formation. Under short-term CO₂ stress, the ratio of 3/6 FA increased and the content of E-16:113 FA in phosphatidylglycerols increased sharply. The drop in protein content especially in the photosystem I (PSI) preparations, as well as diminishing the ratio of F ₇₀₀-to-F ₆₈₆ nm fluorescence (F ₇₀₀/F ₆₈₆) under short-term CO₂ stress argued for the significant damage to PSI. The reversibility of these changes at more prolonged treatment (7 and 10 days) demonstrated that D. salina cells could restore the functional activity of PSI. The lower level of F ₇₀₀/F ₆₈₆, chlorophyll a (Chla)/Chlb, and 3/6 FA ratio in line with the higher level of E-16:113 in the cells growing for a long time at the high CO₂ concentration is characteristic for the new structural and functional state of the photosynthetic apparatus providing for the effective photosynthesis of D. salina under these conditions.