Category Archives: Articles Review

REVIEW of Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating (Bocking et al. 2011 Nat Struct Mol Bio)

Clathrin-dependent vesicular transport is an important pathway in facilitating the movement of modified proteins (ie. post-phosphorylation or post-glycosylation) from the trans-golgi network to other cellular compartments, such as plasma membrane and endosome. Bocking and his group hypothesize that the accumulation of local conformational distortions on the clathrin lattice imposed by Hsc70 leads to the destabilization and uncoating of the clathrin coat. Previous reports have shown that auxilin recruits Hsc70 to bind to the QLMLT motif of clathrin triskelion, followed by uncoating of clathrin coat. However, the exact molecular mechanism of Hsc70 in aiding the uncoating is yet elucidated. As a significant amount of modified proteins are transported out of the golgi network, their work to elucidate the uncoating mechanism of clathrin coat is therefore highly significant.

To approach the question, Bocking group used three key approaches: (i) purified fluorescent clathrin and fluorescent Hsc70 proteins; (ii) formation of clathrin coat with fluorescent clathrin proteins; and (iii) single particle fluorescence imaging assay. Purified fluorescent proteins formed the foundation of their experiments. First, they purified Hsc70, wildtype clathrin and mutant clathrin that lacks the QLMLT motif. Next, they used maleimide to crosslink fluorophore AF568 to Hsc70 and fluorophore AF488 to clathrin proteins. The fluorescent products enabled the group to detect concentrations of each protein at different wavelengths. Second, they made fluorescent clathrin lattice by introducing the accessory protein, AP-2. They also made mixed clathrin coat using different concentration ratios of fluorescent wildtype clathrin to fluorescent mutant clathrin. Last, they designed a single particle fluorescence imaging assay, in which they put antibody against the light chain of clathrin to capture fluorescent clathrin lattice on coverslips. Then they added auxillin, followed by the addition of fluorescent Hsc70 to study the function of Hsc70 in disassembly. To control the disassembly of the coat, they also controlled the stability of lattice at pH6.0, and triggered disassembly at pH6.8.

Using the above approaches, Bocking and his group discovered that there are two major phases of Hsc70-dependent uncoating reaction, including accumulation of Hsc70 and unlocking of lattice, and rapid disassembly. The accumulation of Hsc70 is required prior to disassembly of the clathrin lattice, while the concentration of Hsc70 and the percent of uncoating shows a linear relationship. They also extrapolated that the uncoating is triggered when Hsc70 is accumulated to about 1 molecule for every 2 clathrin triskelions. Furthermore, without the QLMLT Hsc70-binding motif at the C terminal of clathrin, the clathrin coat does not undergo disassembly in the presence of Hsc70. Additionally, the percentage of uncoating depends on the number of wildtype clathrin but not the number of clathrin mutant. Bocking and his group also proposes an alternative function of auxilin. Aside from recruiting Hsc70 to the specific QLMLT domain, auxilin can also recruit Hsc70 to other nonspecific sites. With the presence of both unspecific binding and specific binding of Hsc70 to clathrin, it is estimated that there is one Hsc70 for each clathrin triskelion.

Bocking and his group have provided strong supporting results to validate their hypothesis. Their approach, in particular the design of a single particle fluorescence imaging assay, has effectively justified the contribution of Hsc70 in initiating and disassembling the clathrin coat. Bocking showed the importance of QLMLT motif in initiating the disassembly process by Hsc70 binding. What about the substrate binding domain of Hsc70 that is thought to bind to clathrin? It would be interesting to confirm the importance of Hsc70 by creating a Hsc70 mutant that carry an altered substrate binding domain. Hsc70 mutant can no longer recognize clathrin QLMLT mutant. Thus, Hsc70 mutant should not initiate disassembly, which supports and further confirms their findings. Furthermore, if Hsc70 can be recruited to various nonspecific sites during the accumulation phase, then what makes the specific site (QLMLT) on clathrin so favorable for (1) auxilin to recognize and bind, and (2) Hsc70 to bind? In other words, future research should look into how this specific QLMLT  motif is more favorable than other nonspecific sites. This is important because nonspecific sites may also be a key factor in the disassembly reaction. Despite the initiation of disassembly requires Hsc70 to bind to the specific QLMLT motif, the binding to nonspecific sites might still be useful in catalyzing and maintaining a fast disassembly progress.

References:

Nat Struct Mol Biol. 2011 Mar;18(3):295-301. doi: 10.1038/nsmb.1985. Epub 2011 Jan 30.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating.

REVIEW of Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites (Stefan et al. 2011 Cell)

Phosphoinositides (PI) is an essential signaling molecule as it is a component of a diversified number of cellular functions, such as growth, differentiation, migration, and endocytosis. In this paper, Stefan and his group hypothesized that monophosphorylated PI4P at the PM is linked to Sac1 at the ER with the help from PI4P binding proteins. Their hypothesis is based on the assumption that ER Sac1 controls PM PI4P at the PM/ER membrane contact sites. Previous research reported ∆Sac1 leads to accumulation of PI4P at PM. Sac1 was shown to regulate PI4P at ER and Golgi, however the linkage between the two at the PM was yet investigated. Therefore, this paper is significant by identifying oxysterol-binding homology (Osh) proteins as the missing link connecting Sac1 PI phosphatase activity to PI4P at PM.

To approach the hypothesis, this group utilized three key major experimental procedures: PI analysis, protein-binding assays and phosphatase assays. First, to measure the level of PI4P, PI analysis was conducted. Yeast was harvested and its PI was labeled with myo-[2-3H]-inositol, followed by HPLC separation and radio-detection to quantify PI4P. Next, protein-binding assays were conducted to determine interaction. Proteins with GST-tag was immobilized on sepharose 4B and incubated with potential interactors. The bead was then washed and bound protein was eluted for immunoblotting to identify potential interaction. Crosslinking was also used to isolate indirect complex. Furthermore, phosphatase assay was conducted to measure the phosphate released from the mixture of microsomes and liposomes/PI4P, or proteins and liposomes/PI4P, depending whether the assay is for microsome phosphatase or Sac1 phosphatase. For Sac1 assay, purified proteins was added to liposome/PI4P, then the release of phosphate was measured by addition of Biomoi Green, followed by quantification by a photometer.

This group started the investigation by characterizing the domains of Osh proteins. Osh 1,2,3 are similar to mammalian OSBP which contains a PH domain that bind to PI4P. Osh3 is localized to PM/ER contact sites in response to the level of PI4P at PM. The FFAT motif of Osh3 can rescue the growth defect of Osh∆:CenOshTS. In addition, Osh3 interacts with VAP proteins, Scs2 and Scs22, via FFAT motif. The group continued to look at the connection between Osh proteins and PI4P level. PI4P level was 6-7 fold higher in Osh∆:CenOsh4 and Osh∆:CenOsh4TS from baseline, which indicates that Osh proteins have a regulatory role in PI4P level. This is also consistent with the elevated PI4P level from Osh2∆ Osh3∆ double mutant. Osh∆:CenOsh4TS showed 18 fold increase in PI4P level, suggesting that Osh proteins also regulate the activity of Sac-1. Furthermore, in ∆Scs2, ∆Scs22 double mutant, PI4P level was up by 2.9 fold and PI4P was stabilized at the PM. Microsomes without these VAP proteins showed reduced Sac1 activity and PI4P turnover, which can be rescued by Osh3. This indicated that VAP proteins regulate PM PI4P and Sac1 activity. OSBP-related sterol-binding domain (ORD) of Osh3 and Osh4 was able to activate Sac1. Osh43E (R236E, R242E, R243E) mutant showed impaired ability to bind to PI and stimulate Sac1 activity. Comparing to baseline, PI4P level was 17 fold higher in Osh∆:CenOsh4TS strain coexpressing Osh3E; and ORD mutants of Osh4 also showed 19-21 fold increase in PI4P, indicating that PI and sterol binding are essential for Osh4-mediated PI4P metabolism. Osh7 and Osh3 were found to interact with Sac1 through transient interaction or indirect interaction. Interaction between some Osh proteins and Sac1 is dependent on the interaction between ORD and Sac1.

Stefan and his group had presented a convincing story. I agree that Osh proteins regulate phosphoinositide metabolism at the contact sites between ER and PM. For the future direction, we have to (i) link this finding to a bigger picture, as in how the regulation at PM influences the downstream pathways, and (ii) distinguish PM PI4P from PI4P localized elsewhere. In order to do so, overexpress Osh proteins in cells, followed by immunoblotting for markers of downstream pathways. By establishing downstream expression profiles, we would be able to know what the key pathways are regulated by PM PI4P, but not other PI4P that are localized somewhere else.

Reference:

Cell. 2011 Feb 4;144(3):389-401. doi: 10.1016/j.cell.2010.12.034.

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites.

Review: Anti-apoptotic Mcl-1 protein controls the type of cell death in Theiler’s virus-infected BHK-21 cells

Significance:

•Knockdown of prosurvival Mcl-1 reduces virus titer not by translation or genome replication.
•It may be due to a defect in virion assembly possibly due to capsid protein cleavage from activated caspases (Capsid of feline calicivirus and mink disease virus are cleaved by caspases)

 

Findings in a glance:
•F1: BeAn infected cell mostly go through necrosis, while 20% goes through apoptosis. The release of ROS is significantly increased at 10hpi and 12hpi in BeAn infected cell comparing to mock infected cell.
•F2: They saw that the prosurvival  Mcl-1 is decreased during infection.
*It is strange that F2C and F2D Mcl-1 levels are not the same. It looks like 24hpi from F2C is higher than 12hpi from F2D
•F3: Knockdown of Mcl-1 reduces the BeAn titer.
•F4: They derived stable cell line with lowered Mcl-1. They showed an earlier activation of caspase 9 and caspase 3
•F5: The reduction of titer is not due to translation or genome replication defect, shown by northern blot analysis, pulse-chase
•F6:BeAn infection increases expression of p53, activates p53, Noxa and Bax
•F7: They also saw necrosis in oligodendrocytes.
Reference:

J Virol. 2012 Feb;86(4):1922-9. Epub 2011 Nov 30.

The antiapoptotic protein Mcl-1 controls the type of cell death in Theiler’s virus-infected BHK-21 cells.

Arslan SY, Son KN, Lipton HL.