A search of a sequence database with the sequence of HKLP shows a good match to

Question

A search of a sequence database with the sequence of HKLP shows a good match to a yeast protein. You decide to use yeast genetics to identify other gene products involved in the movement of the vesicles. You make a deletion mutant in yeast of the gene for kinesin-like protein and observe a phenotype (the yeast strain is called YKLP?). You then perform a genetic screen for the same phenotype. This identifies two other distinct genes, YKS1 and YKS2. You engineer each of YKS1, YKS2 and YKLP to be expressed in yeast as a GFP fusion. Each fusion rescues the mutated version of the equivalent wildtype gene. Using these tools, you look at targeting of GFP versions of the other two gene products in mutants of YKLP, YKS1 and YKS2, separately (the mutant yeast strains are called YKLP?, YKS1?, and YKS2?, respectively)

Assuming that the proteins act sequentially, show the order in which the gene products act to localise the YKLP to vesicles, explaining your reasoning

Explanation / Answer

TRANSPORT vesicles mediate the movement of protein cargo between the organelles of the secretory pathway.

The SEC16 gene had previously been shown to be one of the genes required for the formation of ER transport vesicles in vivo (Kaiser and Schekman, 1990). SEC16 interacts genetically with SEC23, SEC13, and SAR1, genes whose products are part of a cytosolic protein coat, termed COPII, that encapsulates vesicles assembled from ER membranes in vitro (Barlowe et al., 1994). Taken together, these results suggested that SEC16 might take part in the formation of the COPII vesicle coat. In this report, Sec16p finds its place as a constituent of COPII-coated vesicles. This conclusion rests on two findings. First, Sec16p appears to be associated with ER-derived vesicles produced in an in vitro budding reaction. When membranes bearing Sec16p are incubated with cytosol, some of the Sec16p is released into a slowly sedimenting fraction in a temperature- and nucleotide-dependent reaction. Release of Secl6p occurs under conditions that closely parallel the behavior of Sec22p, an integral membrane protein marker for ERderived transport vesicles. When the material released from ER membranes is subjected to gel filtration, Secl6p cofractionates with the Sec22p-containing vesicles. These results strongly suggest that Secl6p is coating ER-derived vesicles.  

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