Biology subjects

Hessling, B.

Publications and source records attributed to Hessling, B..

3 recordsLinked to original sources

bioRxiv2019

Nα-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae

SO_SCPLOWUMMARYC_SCPLOWN-terminal (Nt)-acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt-acetyltransferases (Nat) with differing specificities. Nt-acetylation has been implicated in protein quality control but its broad biological significance remains elusive. We investigated the roles of the two major Nats of S. cerevisiae, NatA and NatB, by performing transcriptome, translatome and proteome profiling of natA{Delta} and natB{Delta} mutants. Our results do not support a general role of Nt-acetylation in protein degradation but reveal an unexpected range of Nat-specific phenotypes. NatA is implicated in systemic adaptation control, as natA{Delta} mutants display altered expression of transposons, sub-telomeric genes, pheromone response genes and nuclear genes encoding mitochondrial ribosomal proteins. NatB predominantly affects protein folding, as natB{Delta} mutants accumulate protein aggregates, induce stress responses and display reduced fitness in absence of the ribosome-associated chaperone Ssb. These phenotypic differences indicate that controlling Nat activities may serve to elicit distinct cellular responses.

molecular biology↗

bioRxiv2018

Core Proteome and Architecture of COPI Vesicles

Retrieval of escaped ER-residents and intra-Golgi transport is facilitated by coat protein complex I (COPI)-coated vesicles. Their formation requires the activated small GTPase ADP-ribosylation factor (Arf) and the coat complex coatomer. Here we assess the protein composition of COPI vesicles by combining stable isotope labeling with amino acids in cell culture (SILAC) with in vitro reconstitution of COPI vesicles from semi-intact cells (SIC) using the minimal set of recombinant coat proteins. This approach yields an unbiased picture of the proteome of these carriers. We define a set of ~40 proteins common to COPI vesicles produced from different human as well as murine cell lines. Almost all bona fide COPI vesicle proteins are either ER-Golgi cycling proteins or Golgi-residents, while only a minor portion of secreted proteins was found. Moreover, we have investigated a putative role of {gamma}- and {zeta}-COP as well as Arf isoforms in sorting and recruitment of specific proteins into COPI vesicles. As opposed to the related COPII system, all isoforms of coatomer and all COPI-forming isoforms of the small GTPase Arf produce COPI-coated vesicles with strikingly similar protein compositions. We present a model for the core architecture of COPI vesicles.

biochemistry↗

bioRxiv2018

Proteomic Profiling of Mammalian COPII Vesicles

Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on formation and fusion of vesicular carriers. COPII vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER). They are formed by sequential recruitment of the small GTP binding protein Sar1, the inner coat complex Sec23/24, and the outer coat complex Sec13/31. In order to investigate the roles of mammalian Sec24 isoforms in cargo sorting, we have combined in vitro COPII vesicle reconstitutions with SILAC-based mass spectrometric analysis. This approach enabled us to identify the core proteome of mammalian COPII vesicles. Comparison of the proteomes generated from vesicles with different Sec24 isoforms confirms several established isoform-dependent cargo proteins, and identifies ERGIC1 and CNIH1 as novel Sec24C- and Sec24A-specific cargo proteins, respectively. Proteomic analysis of vesicles reconstituted with a Sec24C mutant, bearing a compromised binding site for the ER-to-Golgi QSNARE Syntaxin5, revealed that the SM/Munc18 protein SCFD1 binds to Syntaxin5 prior to its sorting into COPII vesicles. Furthermore, analysis of Sec24D mutants implicated in the development of a syndromic form of osteogenesis imperfecta showed sorting defects for the three ER-to-Golgi QSNAREs Syntaxin5, GS27, and Bet1.

biochemistry↗