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Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis
Andreas Küberl1, Benjamin Fränzel, Lothar Eggeling
1IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, Jülich, Germany.
Abstract:
In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.
Insights
The prokaryotic ubiquitin-like protein (Pup) modifies proteins in Corynebacterium glutamicum lacking a proteasome. This study identified 55 pupylated proteins, revealing non-proteasomal roles for pupylation.
Area of Science:
- Microbiology
- Molecular Biology
- Proteomics
Background:
- The prokaryotic ubiquitin-like protein (Pup) targets proteins for proteasomal degradation in mycobacteria.
- Some actinobacteria possess Pup but lack a proteasome, suggesting alternative Pup functions.
- Corynebacterium glutamicum is a non-pathogenic model organism relevant for studying Pup in proteasome-lacking bacteria.
Purpose of the Study:
- To investigate pupylation in Corynebacterium glutamicum, a proteasome-lacking actinobacterium.
- To identify pupylated proteins and their modification sites in C. glutamicum.
- To understand the potential roles of pupylation beyond proteasomal degradation.
Main Methods:
- Construction and characterization of a pup deletion mutant (Δpup) in C. glutamicum.
- Expression of a His-tagged Pup derivative for affinity purification of pupylated proteins.
- Proteomic analysis using Multidimensional Protein Identification Technology (MudPIT) and MALDI-TOF-MS/MS.
Main Results:
- Pupylation is dispensable for aerobic growth of C. glutamicum on standard medium.
- 55 pupylated proteins and 66 pupylation sites were identified in C. glutamicum.
- The majority of identified pupylated proteins are involved in metabolism and translation, similar to findings in mycobacteria.
Conclusions:
- This study presents the first comprehensive pupylome of a proteasome-lacking actinobacterium.
- Results demonstrate that pupylated proteins can have fates other than proteasomal degradation.
- Pupylation in C. glutamicum highlights conserved and divergent roles of this post-translational modification in bacteria.
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