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Characterization of MYR1, a dosage suppressor of YPT6 and RIC1 deficient mutants
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2008 (English)In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 53, no 4, 235-47 p.Article in journal (Refereed) Published
Abstract [en]

Membrane traffic is tightly regulated and the Rab protein family of small GTPases plays a central role in this regulation. One member of this family is the Saccharomyces cerevisae protein Ypt6. To search for new genes interacting with Ypt6-related pathways, we performed a genetic screen for high copy suppressors of ypt6Δ temperature sensitivity at 35°C. Among the suppressors, MYR1 was also able to suppress the temperature sensitive mutant lacking Ric1, a subunit of the Ypt6 guanine exchanging factor complex Ric1/Rgp1. Myr1 is characterized by a coiled coil region and a GYF domain, a protein module binding proline-rich sequences. Myr1 is able to bind membranes but is also associated with larger structures insoluble in Triton X-100. By immunofluorescence, Myr1 shows a network-like pattern as well as small foci. Overexpression of Myr1 influences nuclear envelope morphology and high levels are lethal. This lethality is rescued when the N-terminal region, containing the GYF domain, is deleted. The transcription profile of a myr1Δ strain shows effects on genes involved in nuclear migration, Ras signalling and transcription. Taken together, these results suggest that Myr1 is a novel factor linked to the secretory pathway and important cellular regulatory mechanisms.

Place, publisher, year, edition, pages
2008. Vol. 53, no 4, 235-47 p.
Keyword [en]
Ypt6, Ric1, GYF domain, Membrane traffic, Coiled coil, Yeast, Myr1
Identifiers
URN: urn:nbn:se:su:diva-25147DOI: 10.1007/s00294-008-0183-0OAI: oai:DiVA.org:su-25147DiVA: diva2:198998
Note
Part of urn:nbn:se:su:diva-7764Available from: 2008-05-22 Created: 2008-05-22 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Membrane Stress and the Role of GYF Domain Proteins
Open this publication in new window or tab >>Membrane Stress and the Role of GYF Domain Proteins
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.

Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.

We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 71 p.
Keyword
SYH1, SMY2, YPT6, RIC1, MYR1, processing bodies, vesicular trafficking, lipid bilayer, budding yeast, GYF, membrane stress, mRNA decay, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-7764 (URN)978-91-7155-683-7 (ISBN)
Public defence
2008-06-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 14:00
Opponent
Supervisors
Available from: 2008-05-22 Created: 2008-05-22Bibliographically approved
2. Functional studies of nuclear envelope-associated proteins in Saccharomyces cerevisiae
Open this publication in new window or tab >>Functional studies of nuclear envelope-associated proteins in Saccharomyces cerevisiae
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins.

The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics.

In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 58 p.
Keyword
Nucleus, nuclear envelope, nuclear pore complexes, vesicular traffic, arginine methylation, Rmt2, Cwh43, Myr1
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-7599 (URN)978-91-7155-666-0 (ISBN)
Public defence
2008-05-29, MA 636, Södertörns högskola, Alfred Nobels allé 7, Huddinge, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2010-01-13Bibliographically approved

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