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Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40)
1Institut de Biologie, Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS EP 2104, Montpellier, France.
Abstract:
The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.
Insights
The human I-mfa domain-containing protein (HIC) mRNA yields two isoforms, p32 and p40. Researchers identified a nucleolar localization signal (NoLS) in the p40 isoform, directing it to the nucleolus.
Area of Science:
- Molecular Biology
- Cell Biology
- Protein Biochemistry
Background:
- The human I-mfa domain-containing protein (HIC) mRNA generates two isoforms, p32 and p40, via alternative translational initiation.
- These isoforms exhibit distinct subcellular localizations, with p32 in the cytoplasm and p40 in both cytoplasm and nucleolus.
Purpose of the Study:
- To investigate the presence and location of a nucleolar targeting sequence within the N-terminal region of the HIC p40 protein isoform.
- To identify the specific structural motifs responsible for the nucleolar localization of p40.
Main Methods:
- Utilized COS cells transfected with eukaryotic expression vectors encoding GFP-fused p40 N-terminal fragments and deletion mutants.
- Analyzed subcellular localization of fusion proteins using fluorescence microscopy.
- Confirmed the functionality of the identified nucleolar localization signal (NoLS) by fusing the sequence to GFP.
Main Results:
- Fusion proteins of GFP with the p40 N-terminus localized to the nucleolus, suggesting the presence of a nucleolar localization signal (NoLS).
- Deletion mutant analysis pinpointed a 19-amino acid domain containing an arginine-rich subdomain as crucial for nucleolar localization.
- Direct fusion of this 19-amino acid sequence to GFP resulted in its migration into the nucleolus, validating it as an authentic NoLS.
Conclusions:
- The HIC p40 protein isoform contains a functional nucleolar localization signal (NoLS) within its N-terminal 19 amino acids.
- This NoLS is responsible for the observed localization of p40 in the nucleolus.
- The identified NoLS shares characteristics with other known nucleolar localization signals, specifically its arginine-rich subdomain.